Formulations and methods related to eye irritation and related

ABSTRACT

The disclosure relates to formulations and methods for the in vitro testing of ocular irritants. It was discovered that adding an antioxidant formulation to in vitro ocular irritation tests, including for example, a biochemical ocular irritation test, a reconstituted human corneal epithelium (RhCE) ocular irritation test and an excised eye depth of injury (DoI) test, substantially reduces the rate of false positives without diminishing test sensitivity, resulting in more accurately predicting ocular irritancy of test substances. More particularly, the disclosed method employs relatively high physiologic concentrations of one or more antioxidants that are normally present in tears. In a variation, much higher concentrations of one or more antioxidants may provide protection against in vivo exposure to ocular irritants.

RELATED APPLICATIONS

The present patent document is a divisional of application Ser. No.17/203,467, filed Mar. 16, 2021, which claims the benefit of the filingdate under 35 U.S.C. § 119(e) of Provisional U.S. Patent ApplicationSer. No. 63/048,112 filed Sep. 4, 2020. All of the foregoingapplications are hereby incorporated by reference.

BACKGROUND

Most chemical safety testing for the eye has traditionally beenperformed using the method of Draize (Draize et al., 1944), as modifiedby Kay and Calandra (Kay and Calandra, 1962). This now-controversialprocedure involves instilling the substance under evaluation within theconjunctival sac of a New Zealand White rabbit. Indices of toxicity arerecorded for the cornea, iris, and conjunctiva at regular time intervalsfor up to 21 days.

Draize test data have traditionally been used to derive a numericalscore of ocular irritation; however, modern classification systems usethe same data with a slightly different statistical treatment to developan irritation category. Modern ocular irritation classification schemesinclude the European Union (EU), Globally Harmonized System ofclassification and labeling of chemicals (GHS), and EnvironmentalProtection Agency (EPA) systems, which are not harmonized with oneanother. The EU Dangerous Substance Directive (DSD) classification andlabeling system does not include cosmetics; it was applied in accordancewith the Commission Directive 2001/59/EC and includes categories R41(risk of serious damage to the eye) and R36 (ocular irritant) (EC,2001). DSD was replaced by the Classification, Labeling, and Packaging(CLP) regulation aligned with GHS (EC, 2008a). The GHS system includesclasses NC (not classified as an irritant), 2A (reversal by 7 days), 2B(reversal by 14 days), and 1 (no reversal by 21 days) (EC, 2008b; UN,2011). GHS classification is used to satisfy U.S. Food and DrugAdministration and international safety labeling requirements and playsan important role in commercial product liability and consumer productsatisfaction. Guidance documents produced by the Organization forEconomic Trade and Development (OECD) are available to coordinateinternational trade. The OECD describes the standard rabbit eye test(the gold standard for GHS eye safety classification), which is requiredfor safety data sheet documentation accompanying hazardous chemicals andproducts.

The EPA classification includes classes I (corrosive), II (moderateirritant), III (mild irritant), and IV (nonirritant), in accordance withthe guidelines in the Label Review Manual (US EPA, 2003) and based ontest methods described in the Acute Eye Irritation Health Effects TestGuideline (US EPA, 1998). Corrosives result in irreversible damage tothe eye, whereas ocular irritation is reversible. EPA class III includesirritation at 24 h. The GHS classification system, now widely acceptedin the EU, does not include a category with a comparable short-durationsensitivity limitation. The EPA classification system is required foragrochemicals and other registrations and has commercial significance,especially for cosmetics and personal care products used around the eye.

One of the needs for nonanimal safety tests originated from bans orpending bans on the use of animals for the safety of cosmetics and otherproducts. The EU banned animal testing of finished cosmetic products in2004, animal-tested ingredients 4 years later, and the transport andsale of cosmetics containing ingredients tested on animals in 2013,pledging to push other parts of the world to accept alternatives(Kanter, 2017). As of 2014, there are bans or severe limitations inNorway, Israel, India, and Brazil (Senate Joint Resolution 22, 2014),and by 2017, the list of countries had grown to 37, according to theHumane Society of the U.S. (Humane Society, 2017).

The United States has been slow to ban animal testing or mandate the useof nonanimal alternatives in the product testing industry; however,recent legislation will ban animals for a wide range of testingapplications that have traditionally used live animals. Bill H.R.2790“The Humane Cosmetics Act” was introduced on Jun. 6, 2017 and wouldprohibit animal testing of cosmetics within 1 year and the sale ortransport of cosmetics tested on animals within 3 years after enactment,which is now supported by more than 200 cosmetics companies andstakeholders (H.R.4148, 2014). Additionally, the “Frank R. LautenbergChemical Safety for the 21st Century Act”—S.697, which revises the ToxicSubstances Control Act of 1976 (TSCA)—was passed on Jun. 22, 2016. TheTSCA now requires EPA to evaluate existing and new chemicals todetermine whether regulatory control of a certain chemical is warrantedand if it presents an unreasonable risk of injury to health or theenvironment so as to reduce risks to a reasonable level. The law alsorequires EPA to “reduce and replace, to the extent practical . . . theuse of vertebrate animals in testing chemicals to provide information ofequivalent or better scientific quality and relevance for assessingrisks of injury to health or the environment of chemical substances ormixtures . . . ” and to develop a strategic plan within 2 years ofenactment or by June 2018 (S.697, 2016). Section 4 of the new lawincludes specific guidance on the use of nonanimal tests when availablefor initial screening and tiered testing of chemical substances andmixtures (S.697, 2016). Therefore, an accurate and internationallyaccepted nonanimal test for ocular irritation is needed.

In light of these issues, increased interest has focused on thedevelopment of nonanimal testing methods and strategies to replace theDraize live animal eye irritation test. Toward this end, the InteragencyCoordinating Committee for the Validation of Alternative Methods(ICCVAM) and the European Centre for Validation of Alternative Methods(ECVAM) conducted retrospective evaluations of data available for fourorganotypic methods and four cytotoxicity and cell function testmethods. Based on these retrospective evaluations, the predictiveperformance of all individual test methods was not felt to be sufficientfor any one test, or group of tests, to fully replace the rabbit Draizeeye test (ICCVAM, 2009). ICCVAM and ECVAM did, however, accept thebovine cornea opacity and permeability (“BCOP”) test, isolated chickeneye test, cytosensor microphysiometer (CM, for water-soluble materials),and fluorescein leakage test (for water-soluble materials) as screeningtests for the identification of materials classified as NC, ocularcorrosives, and severe eye irritants, and the CM as a screening test forthe identification of materials classified as NC (surfactants andsurfactant mixtures). Recently, differentiated cell culture models,including the EPIOCULAR eye irritation test, the SKINETHIC human cornealepithelium, and the LABCYTE CORNEA-MODEL24, were demonstrated to haveutility for the detection of NC (OECD, 2019a).

No single nonanimal test, or combination of nonanimal tests, cancurrently detect GHS-classified reversible irritation with any degree ofstatistical certainty (Wilson et al., 2015).

Overall, there are a limited number of types of ocular irritation teststhat do not require the use of animals. These tests include cellculture-based tests, tests based on excised animal eyes, egg-basedtests, and biochemical tests. All of these tests fail to identify ormodel some essential component of the live eye and have either poorspecificity or sensitivity. The lack of understanding of the underlyingreasons why some substances are much more damaging than others hashindered the development of nonanimal tests for eye safety testing. Allsensitive tests for ocular irritation have a high false-positive rate(about 40%). Those familiar with the state of the art say the high falsepositive rates of nonanimal tests is because nonanimal tests are onlyable to measure initial damage, but do not accurately model therepair/reversibility of the damage.

False negatives are dangerous because the nonanimal test predicts that achemical or product is safe for the eye, when in fact, the substanceirritates or corrodes the live eye. False positives are dangerousbecause people do not believe test methods with a high false positiverate resulting in ignoring warning labels, and manufacturers are slow toadopt methods with a high false positive rate because they erroneouslyrestrict the use of safe products and scare away consumers.

SUMMARY

An in vitro method for predicting ocular irritancy of a test substanceis disclosed. The method includes applying the test substance to an invitro irritancy test system in the presence of an antioxidantformulation under conditions in which antioxidant is allowed to interactwith the test substance, including where the antioxidant formulation is:(1) mixed with the test substance prior to applying to the test system,(2) added to the test system prior to applying the test substance, (3)or both (1) and (2); measuring a test system response; and predictingthe ocular irritancy of the test substance based on the test systemresponse.

In some embodiments of the method, the antioxidant formulation comprisesone or more compounds selected from ascorbic acid, baicalein,beta-carotene, bilirubin, caeruloplasmin, catechin, cobalamin, coenzymeQ10, cortisone, cryptoxanthin, crystallin, curcumin, cyanidin,delphinidin, epigallocatechin-3-gallate, esculetin, estradiol, estriol,folic acid, genistein, glutathione, glutathione peroxidase, human serumalbumin, idebenone, kaempferol, L-acetylcarnitine, L-cysteine, lipoicacid, L-tyrosine, lutein, lycopene, melatonin, mexidol, myo-inositol,myricetin, N-acetyl cysteine, estrogen, omega-3, omega-6, omega-9,pelargonidin, peonidin, petunidin, piceatannol, pigment epitheliumderived factor, quercetin, resveratrol, riboflavin, selenium, silymarin,superoxide dismutase, taurine, tempol, thiamine, thioredoxin,thymoquinone, transferrin, ubiquinol-10, uric acid, vitamin A, vitaminD3, vitamin E, and zeaxanthin.

In some embodiments, the antioxidant formulation comprises about 0.27-60mM ascorbic acid.

In some embodiments, the test system is a biochemical test systemcomprising purified or semi-purified molecules. In other embodiments,the test system comprises reconstituted human corneal epithelium (RhCE).In other embodiments, the test system is a Depth of Injury (DoI) testsystem comprising excised eyes.

In one embodiment, the test system is selected from the OPTISAFE ocularirritation test, the IRRITECTION ocular irritation test, the EPIOCULARocular irritation test, the SKINETHIC ocular irritation test, theLABCYTE CORNEA-MODEL24 ocular irritation test, the MCTT HCE™ ocularirritation test, the Short Time Exposure ocular irritation test, theHET-CAM ocular irritation test, the CAMVA ocular irritation test and theDEPTH OF INJURY (DoI) ocular irritation test.

A method for reducing false positive rates of nonanimal eye irritationtests is disclosed. The method includes: overlaying an antioxidantformulation onto a surface of a differentiated eye tissue, comprisingreconstituted human corneal epithelium or excised eye tissue; adding atest substance to the antioxidant formulation on the surface of thedifferentiated eye tissue; exposing the differentiated eye tissue to thetest substance for a first period of time; washing the surface of thedifferentiated eye tissue with a buffered salt solution to remove thetest substance; after a second period of time, measuring cell viabilityof the differentiated eye tissue; and relating the measured cellviability to an index of irritation, which can be categorized accordingto established ocular irritancy classes, where the false positive rateis reduced compared to performing the method without overlaying with theantioxidant formulation.

In some embodiments of the method for reducing false positive rates, theestablished ocular irritancy classes are selected from a nonirritant, aminimal irritant, a mild irritant, and a severe irritant. In otherembodiments, the established ocular irritancy classes include GHScategories NC, 2, 2B, 2A, and 1, or EPA categories IV, III, II, and I.

In some embodiments, the antioxidant formulation comprises 1.70 mM (0.3mg/ml) ascorbic acid, 1% bovine serum albumin and 5% dextran in abuffered saline solution.

In some embodiments, the washing step further comprises a subsequentwash with additional antioxidant formulation.

An antioxidant formulation is disclosed. The formulation includesascorbic acid at a concentration of about 0.27-60 mM, serum albumin at aconcentration of about 0.05% to 10%, and dextran at a concentration ofabout 3% to 30%, in a buffered saline solution.

In one embodiment, the ascorbic acid has a concentration of about 1.70mM (0.3 mg/ml).

In one embodiment, the serum albumin is bovine serum albumin at aconcentration of 1%.

In one embodiment, the dextran has a concentration of 5%.

In one embodiment, the buffered saline solution includes about 6 mg/mlNaCl in a HEPES buffer

In some embodiments, the formulation reduces the false positive rate ofin vitro nonanimal eye irritation tests.

In some embodiments, the formulation reduces the damage caused byexposure to eye irritants in vivo. In a particular embodiment, thepost-exposure protective formulation includes ascorbic acid at aconcentration of 17.0 mM (3 mg/ml).

A procedure and reagent are disclosed for the accurate prediction of eyetoxicity after a chemical, product, or material exposure in whichantioxidants that model those found in the live eye are added, and theaddition of the antioxidants results in a lower FP rate, as determinedby comparing the nonanimal test method FP rate to the live animal orhuman TN rate.

In some embodiments, known irritants may include one of more of thefollowing: dodecanaminium,N-(2-hydroxy-3-sulfopropyl)-N,N-dimethyl-,1-naphthaleneacetic acid,1-octanol, 1,2,4-triazole, sodium salt, 1,3-di-isopropylbenzene,1,3-diiminobenz (f)-isoindoline, 1,5-hexadiene, 2-benzyl-4-chlorophenol,2-benzyloxyethanol, 2-ethoxyethyl acetate (cellosolve acetate),2-ethyl-1-hexanol, 2-hydroxyisobutyric acid ethylester,2-hydroxyisobutyric acid, 2-methyl-1-pentanol, 2-methylbutyric acid,2-naphthalene sulfonic acid, formaldehyde, hydroxymethylbenzene sulfonicacid monosodium salt, 2-nitro-4-thiocyanoaniline,2,2-dimethyl-3-pentanol, 2,2-dimethyl butanoic acid,2,5-dimethyl-2,5-hexanediol, 2,6-dichlorobenzoyl chloride,2,6-dichloro-5-fluoro-beta-oxo-3-pyridinepropanoate,3-chloropropionitrile, 3,3-dithiodipropionic acid, 3,4-dichlorophenylisocyanate, 4-(1,1,3,3-tetramethylbutyl)phenol, 4-tert-butylcatechol,4-carboxybenzaldehyde, 4-chloro-methanilic acid, 6-methyl purine,p-tert-butylphenol, acetic acid, acetone, acid blue 40, acid red 92,alpha-ketoglutaric acid alpha, ammonia, aluminum chloride,gamma-aminopropyltriethoxy silane, ammonium nitrate, antimony oxide,benzalkonium chloride, benzalkonium chloride (10%), benzenesulfonylchloride, benzethonium chloride (10%), benzene, 1,1′-oxybis-,tetrapropylene derivatives, sulfonated, sodium salts, benzotrichloride,benzyl alcohol, beta-resorcylic acid, bis-(3-aminopropyl) tetramethyldisiloxane, butanol, butyl acetate, butyl cellosolve, butyl dipropasolsolvent, butylnaphthalene sulfonic acid sodium salt, butyrolactone,calcium thioglycolate, captan 90-concentrate (solid), camphene,cetylpyridinium bromide (10%), cetylpyridinium chloride (10%),cetyltrimethylammonium bromide (10%), chlorhexidine, chloroform,cyclohexanol, cyclohexanone, cyclohexyl isocyanate, cyclopentanol,deoxycholic acid sodium salt (10%), di(2-ethylhexyl) sodiumsulfosuccinate (10%), di(propylene glycol) propyl ether,dibenzoyl-L-tartaric acid, dibenzyl phosphate,diethylaminopropionitrile, domiphen bromide (10%), ethanol, ethyl2-methyl acetoacetate, ethyl trimethyl acetate, glycidyl methacrylate,granuform, hydroxyethyl acrylate, imidazole, isobutanal, isobutylalcohol, isopropyl alcohol, lactic acid, lauric acid,lauryldimethylamine oxide, lime, m-phenylene diamine, magnesiumhydroxide, maneb, methoxyethyl acrylate, methyl acetate, methylcyanoacetate, methyl cyclopentane, methyl ethyl ketone (2-butanone),methyl isobutyl ketone, methylpentynol, methylthioglycolate, myristylalcohol, n-acetyl-methionine, n-butanol, n-hexanol, n-laurylsarcosinesodium salt (10%), n-octylamine, N,N,N′,N′-tetramethylhexanediamine,naphthalenesulfonic acid, 2-naphthalenesulfonic acid, sodium salt,nitric acid, organofunctional silane 45-49, phosphorodicloridic acid,hydrogenated tallow amine, polyoxyethylene(23) lauryl ether, potassiumlaurate (10%), potassium oleate, promethazine hydrochloride, potassiumhydroxide, protectol PP, pyridine, benzyl-C12-16-alkyldimethyl, silvernitrate, sodium 2-naphthalenesulfonate, sodium hydrogen difluoride,sodium hydrogen sulfate, sodium hydroxide (10%), sodium lauryl sulfate,sodium lauryl sulfate (15%), sodium monochloroacetate, sodium oxalate,sodium perborate tetrahydrate, sodium polyoxyethylene(3) lauryl ethersulfate, sodium salicylate, stearyltrimethylammonium chloride, sulfuricacid, tetra-N-octylammonium bromide, tetraethylene glycol diacrylate,tetrahydrofuran, trichloroacetic acid (30%), trichloroacetyl chloride,triethanolamine, triethanolamine polyoxyethylene(3.0) lauryl ethersulfate, triton X-100, triton X-100 (5%), triton X-100 (10%).

In some embodiments, known nonirritants may include one or more of thefollowing: 1-bromo-4-chlorobutane, styrene, 1,9-decadiene, 2-ethylhexylp-dimethylamino benzoate, 2-methylpentane, 2-(n-dodecylthio)-ethanol,2,2-dimethyl-3-pentanol, 2,4-difluoronitrobenzene, 2,4-pentanediol,3-methoxy-1,2-propanediol, 3-methylhexane, 3,3-dimethylpentane, acrylicacid homopolymer sodium salt, di-n-propyl disulphide, diisobutyl ketone,ethylhexyl salicylate, glycerol, iso-octyl acrylate, isopropyl bromide,isopropyl myristate, iso-octylthioglycolate, methyl trimethyl acetate,n-hexyl bromide, n-octyl bromide, n,n-dimethylguanidine sulfate,polyethylene glycol 400, polyethyleneglycol monolaurate (10 E.O.),polyoxyethylene hydrogenated castor oil (60E.O.), polyoxyethylene(14)tribenzylated phenyl ether, polyoxyethylene(160) sorbitantriisostearate, polyoxyethylene (40) hydrogenated castor oil, potassiumtetrafluoroborate, propylene glycol, sodium lauryl sulfate (3%),sorbitan monolaurate, tetra-aminopyrimidine sulfate, toluene, tritonX-100 (1%), and tween 80.

In some embodiments, antioxidants may include one of more of thefollowing: apigenin, ascorbic acid (about 0.27-60 mM), baicalein,beta-carotene: 0.5-50 μM, bilirubin, caeruloplasmin, catalase (0.6-60μM), catechin, cobalamin, coenzyme Q10, cortisone, cryptoxanthin,crystallin, curcumin, cyanidin (0.01-50 PM), delphinidin (0.01-50 μM),epigallocatechin-3-gallate, esculetin, estradiol, estriol, folic acid,genistein, glutathione (1.0-107 μM), glutathione peroxidase, human serumalbumin, idebenone, kaempferol, L-acetylcarnitine, lipoic acid,L-tyrosine (0.5-45 μM), lutein (0.5-50 μM), lycopene, melatonin,mexidol, myo-inositol, myrecitin, N-acetyl cysteine, estrogen, omega-3,omega-6, omega-9, pelargonidin (0.01-50 μM), peonidin (0.01-50 μM),petunidin (0.01-50 μM), piceatannol, pigment epithelium derived factor(0.8-80 PM, quercetin, resveratrol, riboflavin, selenium (0.2-20 μM),silymarin, superoxide dismutase (1.0-100 μM), taurine (0.2-22.6 μM),tempol, thiamine, thioredoxin, thymoquinone, transferrin (0.08-80 μM),ubiquinol-10, uric acid (0.4-43 μM), vitamin A (1.7-172.7 μg/mL),vitamin D3, vitamin E (0.5-50 μM), and zeaxanthin (0.5-50 μM).

As used herein, “toxicity” is used to refer to a substance's ability todamage, irritate, or otherwise negatively affect an eye. Toxicity may beevidenced by pain, irritation, swelling, opaqueness, redness, anddischarge. Such effects may be temporary or permanent. Accordingly, theword “toxicity” is defined broadly to include any discomfort orunfavorable experience associated with the presence of a substancecontacting an eye. As used herein, “irritancy” or “irritant” is usedbroadly to cover the spectrum of between nonirritating (nontoxic) tohighly corrosive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts control antioxidant titrations. Positive control=2A.Cyclohexanol CASRN 108-93-0. Negative controls=2B. 2,4-Pentanediol CASRN625-69-4 and 2C. Dodecane CASRN 112-40-3. False-positive controls=2D.Triphenyl phosphite CASRN 101-02-0, 2E. Ethyl acetate CASRN 141-78-6,2F. 2,4-Pentanedione CASRN 123-54-6, and 2G. 2,2-Dimethyl-3-pentanolCASRN 3970-62-5. The dashed line shows the GHS NC cut-off.

FIG. 2 depicts reactive oxygen species (ROS) and crosslinker (CL)antioxidant titrations. ROS chemicals (previously classified as falsepositives)=3A. [2-(2-Ethoxyethoxy) ethanol CASRN 111-90-0, 3B.Triethylene glycol CASRN 112-27-6, 3C. Ethylene glycol diethyl etherCASRN 629-14-1, and 3D. Styrene CASRN 100-42-5. Cross-linkers(previously predicted as false positives)=3E. (1,9-Decadiene CASRN1647-16-1 and 3F. 2-Ethoxyethyl methacrylate CASRN 2370-63-0). Thedashed line shows the GHS NC cut-off.

FIG. 3 depicts results from the modified EPIOCULAR reconstituted humancorneal epithelium test system in which antioxidant formulation wasadded. Results from exposure to test substance (styrene) under twoconditions, with or without antioxidant formulation (including 1.70 mM(0.3 mg/ml) ascorbic acid in a buffered salt solution) overlayed on theapical surface of the eye epithelial test model prior to addition of thestyrene (in this case, the GHS NC chemical styrene).

FIG. 4 depicts results from the modified EPIOCULAR reconstituted humancorneal epithelium test system in which a high concentration (17 mMascorbic acid) antioxidant formulation was added.

DETAILED DESCRIPTION

As disclosed herein, the inventors have discovered that high falsepositive rates exhibited by current nonanimal ocular irritancy tests canbe substantially reduced and/or prevented by adding antioxidants, inparticular those found in tears, to in vitro test systems. The highfalse positive rates of current nonanimal tests are likely not onlybecause of a failure to reverse damage caused by irritants after it hasoccurred, but also by a failure of nonanimal tests to model otheraspects of the live eye, including the antioxidant properties of tears,which theoretically prevent initial damage by inactivating reactivemolecules before they have a chance to damage ocular tissue. Asdescribed below, although nonanimal eye tests have been in developmentfor over 25 years, the prevention of chemical damage by modeling tearrelated antioxidant activity has not been a consideration for testdevelopment or strategy to reduce the false positive rate. Indeed,conventional wisdom has led the skilled artisan away from usingantioxidants in nonanimal test systems, because these tests have beenpurposely biased to maximize sensitivity, such that the reduction ofmeasured damage by inactivating reactive molecules has been viewed asreducing the sensitivity of the test method. Nonetheless, as disclosedherein, the addition of live eye related antioxidants specifically andsubstantially reduced the false positive rate, without reducingsensitivity (i.e., no increase in false negatives). Consequently, thespecific and substantial reduction in false positive rate is animportant and unexpected finding.

Cell culture-based tests typically involve applying the test substanceto epithelial tissues grown in a dish to determine its toxicity based onthe degree of cells killed by the substance after a fixed time. Thesetests only examine the outermost layer of the eye (the epithelium).These cells are either skin cells or genetically modified cells that donot contain the antioxidants or chemical composition of tears or thecorneal stroma. As shown in Table 1 below, common cell culture mediaeither does not include antioxidants, or as in the case of MEM mediumused for the BCOP test, contains a concentration of antioxidant(ascorbic acid) far lower than that found in tears. Further, as detailedbelow, antioxidant is not added to the test substance or included duringthe time the test substance is exposed to the cultures.

TABLE 1 Media Used for Cell Culture, Differentiated Tissue and OrganCulture Tests Ascorbic Acid Media Aqueous Media (Y/N) ConcentrationHumor^(1,2) Tear³ EMEM N none 0.81 mM 1.3 mM ATCC 30-2003 (short timeexposure; STE Test MEDIA) DMEM N none 0.81 mM 1.3 mM High Glucose [+]Pyruvate [+] Phenol Red Cat# 11995 (used for tissue and cell culturetests) MEM Alpha Y 0.17 mM 0.81 mM 1.3 mM [+] Nucleosides [−] Phenol Red(USED FOR BCOP) DMEM 2902 N none 0.81 mM 1.3 mM Low glucose [−] phenolred (used for tissue and cell culture tests) ¹de Berardinis et al.,1965. ²https://emedicine.medscape.com/article/2088649-overview ³Patersonand O'Rourke, 1987.

Tests based on fertilized eggs, or the HET-CAM, measure changes to thevessels that extend from the developing yolk to the air cell within theegg; this primitive respiratory tissue (CAM) is a system that is verydifferent from the eye, does not measure antioxidant effects on the eye,and is a visual test that does not detect transparency changes. Noantioxidants are added to this test system.

Organotypic tests using cow, chicken, rabbit, human or pig eyes measurechanges in opacity and permeability (e.g., BCOP test) or fluoresceinretention and/or cornea thickness (e.g., ICE test, PORCORA etc), depthof injury and viability (DoI test and other organotypic tests). Excisedeyes may or may not be metabolically active and may contain someresidual antioxidant activity within the tissue. However, any suchresidual antioxidant activity is much lower than that found in tears,and no tear-like supplement or antioxidant is added at the same time asthe test substance to mimic the tearing process and process of tear andantioxidant mixing with the test substance prior to interacting withtissues.

In addition, these tests use typically use an off the shelf tissueculture medium that may include some low level of antioxidant (below thelevels found in tears as explained above), however, these tissues arefed from the bottom (endothelial side) and the test chemical is appliedto the outermost region of the eye (epithelial side). Therefore, the lowlevels of antioxidants in the medium are in the wrong location tointeract with the chemical when it is applied to the epithelial side,and the reaction kinetics of reactive oxygen species tissue damage areextremely fast, to be effective, the antioxidant mix must form a layerbetween the tissue and the chemical, as in tear film. Therefore, lowlevels of antioxidants in tissue culture medium do not reduce the falsepositive rates of these tests. These tests exhibit high false-positive(FP) rates that do not accurately approximate the live animal responseto the same chemicals indicating they are missing a variable required tocontrol the false positive rate.

Although the nonanimal alternative eye toxicity tests described abovehave been performed for many years, toxicologists and those skilled inthe art do not know all of the underlying reasons why some substancescause persistent or permanent damage to the live eye, while damagecaused by other substances is repaired quickly; and why the availablenonanimal tests all appear to overpredict the toxicity in severalclasses of chemicals (and up until now, these classes, includingoxidants, have not been recognized) compared to live animal or human eyetest results. Nonetheless, modern toxicity classification for labelingand safety data sheets, as well as for other uses, depends on the timeuntil recovery to classify a substance as toxic or nontoxic to the eye;predicting whether the live eye can repair or prevent lesions iscritical to understanding toxicity and provides a regulatoryclassification for a chemical or product.

The Unique Redox Environment of the Eye

The cornea consists of a stroma that is protected by a 5-7-layer thickcorneal epithelium. This epithelium is stratified, nonkeratinizedsquamous tissue. The conjunctiva is composed of 3-5 cell layers ofstratified, nonkeratinized cells. The cornea and conjunctiva function asbarriers to protect the eye from exposure to environmental insultsincluding foreign bodies, microbes, and irritating chemicals.

Tear film consists of three layers-mucin, aqueous, and lipid (inner toouter) that contribute to the health and maintenance of the ocularsurface (Conrady et al., 2016). Lacrimal glands produce the aqueouslayer of the tear film, which is produced at a basal rate of up to 2microliter per minute (Kim et al., 2019) and up to 100-fold higher inresponse to mechanical, thermal, or chemical exposure (reflex rate).These increased aqueous tears dilute, clear, and detoxify chemicals(discussed in detail below).

The human eye contains the mucosal surface of body that is most exposedto the surrounding environment, including atmospheric oxygen, toxicchemicals, and radiation/ROS produced in situ from light-inducedoxidative damage. Current nonanimal tests do not include this mucosa,lacrimal gland, or model tears, which is the first biological fluid tointeract with chemicals that contact the eye. A significant effect onocular surface inflammation, corneal epithelium lesions, and oculardiscomfort is related to dry eye and increased tear film osmolarity. Thetear film has significant levels of antioxidants.

In particular, the cornea is protected against reactive oxygen species(ROS) that include superoxide anion (O₂ ⁻), hydrogen peroxide (H₂O₂),hydroxyl radical (HO*), hydroperoxides (ROOH), peroxyl radicals (ROO*),and singlet oxygen (O₂) (Nita and Grzybowski, 2016; Ung et al., 2017).Cellular phospholipid bilayers are susceptible to ROS-induced damage vialipid peroxidation, which occurs when free radical species includingoxyl radicals, peroxyl radicals, and hydroxyl radicals remove electronsfrom lipids and subsequently produce reactive intermediates that cancause massive damage via redox cycling (Njie-Mbye et al., 2013;Babizhayev, 2016; Tangvarasittichai, 2018; Su et al., 2019). Theoxidation of nucleotides and proteins may lead to changes in geneexpression, mutations, and the formation of insoluble proteinaggregates.

Importantly, the eye is protected against oxidative stress byantioxidants in the cornea, aqueous humor, and tear film. As shown inTable 2, human tear film and aqueous humor have a similar concentrationof antioxidants. Tear film is the first biological fluid to interactwith and potentially detoxify chemicals that contact the eye.Nonetheless, nonanimal tests do not model tears or the tearing process.Likewise, the aqueous humor is continuously generated and drained andhas a composition similar to tear film (Chen, et al; 2009). Aqueoushumor production and turnover is a dynamic process, which like tearing,is not modeled by nonanimal tests. In human tears, ascorbic acid anduric acid account for approximately 50% of the total antioxidantactivity, with ascorbic acid being the most abundant. Other smallmolecules, including reduced glutathione, L-cysteine, and L-tyrosine,make up the rest. Enzymes of the aqueous humor include superoxidedismutase (SOD), which has an activity around 3.5 U/mL (Behndig et al.,1998). In the order of abundance in human aqueous humor, nonenzymaticantioxidants include ascorbic acid (530 WM), L-tyrosine (78 WM), uricacid (43 μM), L-cysteine (14.3 μM), and glutathione (5.5 μM). SODactivity is not believed to contribute significantly to the antioxidantdefense mechanisms of the aqueous humor (Chen et al., 2009).

TABLE 2 Antioxidants Present in Aqueous Humor and Tear AntioxidantAqueous Humor^(a) Tear Film^(a) L-Tyrosine 78 μM 45 μM Uric acid 43 μM328 μM Ascorbic acid 530 μM 665 μM L-Cysteine 14.3 μM 48 μM Glutathione5.5 μM 107 μM Superoxide ~5.1 U/mL^(b) 3.5 U/mL^(b) dismutase ^(a)Chenet al., 2009 ^(b)Behndig et al., 1998

Even though scientists in the area of ophthalmology have characterizedthe importance of protective antioxidant effects on the eye in disease,current in vitro eye tests are theoretically limited in their ability tomodel the response to oxidative stress. Based on our literature review,current nonanimal eye irritation tests have not specifically accountedfor the antioxidant capabilities and properties of the eye; especiallythe tear response that occurs when chemicals first contact the ocularsurface.

Current nonanimal eye irritation tests are highly simplifiedreductionist models without important factors, such as tear film,innervation, and immune responses, that are normally present in the liveeye.

Addition of Antioxidants to OPTISAFE In Vitro Ocular Irritation Test

To address the need for a better and more predictive nonanimal ocularirritation test, we have been developing and improving the chemicallybased, in vitro ocular irritation test, referred to as OPTISAFE. Thistest can discriminate nonirritants from irritants/corrosives in fewerthan 24 h, with only an hour of hands-on time. Furthermore, multipletest samples can be evaluated simultaneously using standard laboratorytechniques and equipment with a shelf life of at least 1 year. Recently,results from a validation study showed that OPTISAFE has a very highsensitivity (100%, the only nonanimal/in vitro test with a zero falsenegative rate) for nonirritant detection and provides a rapid,high-throughput screening method for nonirritants; however, the FP ratewas about 40%, which is similar to other available nonanimal eye safetytests.

Since we have noted that in vitro tests, including OPTISAFE, overpredictmany of the same chemicals (Lebrun et al., 2020), we questioned whetherthese false positives might have common chemical reactions that involvethe formation of reactive oxygen species (ROS).

We complied a new list that compared overpredictions for eye irritationand toxicity by all the different nonanimal tests. We noticed thatnonanimal tests overpredicted the same chemicals that had similarchemical properties. Interestingly, many of these FPs were associatedwith a specific class of materials, ether-alkoxides or chemicalsassociated with oxidative chemistry, which cause oxidative damage andgenerate ROS. This is quite unexpected because oxidative damage and ROSare primary mechanisms of damage to the eye, so it is not expected thatthere would be a need to control this mechanism of damage to the eyewith antioxidants. However, we noticed that a number of oxidants are infact nonirritants; they are either not toxic or damage is prevented bythe live eye. These chemicals became the target for our formulationimprovements and resulted in the discovery described here: therequirement to model the antioxidant capacity of the eye in order to notoverpredict the toxicity of safe chemicals.

Previous studies have shown that the eye expresses high levels ofantioxidants. The eye is exposed to oxidative stress and has mechanismsto defend against ROS on the ocular surface that includes antioxidantsin the cornea, aqueous humor, and tear film. The level of antioxidantintake and plasma levels can have effects on eye disease in humans(Cabrera and Chihuailaf, 2011; Umapathy, et al.; 2013).

Discovery began with the inclusion of various antioxidant mixtures intothe formulation of the OPTISAFE nonanimal eye irritation test. Theantioxidant mixture contains one or more of the following: Glutathione(GSH) (1-10,000 μM), L-cysteine (0.5-5,000 μM), L-tyrosine (0.5-5,000μM), ascorbic acid (3.0-1,000 mM), and/or uric acid (3.0-30,000 μM).True-negative (TN) and true-positive (TP) chemicals were tested inparallel to ensure that effects of the antioxidant mixtures did notalter the results of the other varying mechanism measured. Duringdevelopment, octanol CASRN 111-87-5 and 2,4-pentanediol CASRN 625-69-4were used as TP and TN reference standards. The results indicated thatthe addition of the antioxidant mixture, at any concentration, did notimpact the generated scores of the TP and TN.

Methods

The OPTISAFE method was conducted as previously described (Choksi etal., 2020). Briefly, samples were initially evaluated for solubility,pH, and foaming using a standardized procedure. Based on the outcomes, adecision tree was followed to allow for standardized proceduralmodifications for substances with the following properties: 1) extremepH, 2) insolubility, and 3) categorized as a surfactant. The procedurediffered for materials with these properties. For substances with anextreme pH, the buffering power was evaluated, and standards wereadjusted to match. Insoluble materials were floated instead of placed onmembrane discs. Surfactants were diluted. Materials were tested forpotential to damage to water-soluble or -insoluble macromolecules.Samples were titrated at five dilutions; after incubation, the resultingOD and pH values were compared with quality controls and a standardcurve. In some cases (e.g., the OD exceeds the photometric limit of thespectrophotometer or an inverse dose-response curve), no result could beprovided (“Criteria Not Met” abbreviated CNM).

Antioxidant Screening

A screen of the six major abundant antioxidants present in aqueous humorand tear film as listed in Table 2 was performed. In some cases,evaluated materials were added to the OPTISAFE formulation, in othercases the antioxidant can be added to the test matrix or to the testsubstance, and/or overlayed onto test system prior to dosing the testsubstance on the test system. In certain embodiments, the first thingthe test substance interacts with is preferably the antioxidant. Forinstance, in the OPTISAFE test, this interaction between test substanceand antioxidant occurs in the assay matrix, or optionally, as anoverlay. In cell and organotypic eye irritation assays, the testsubstance can be premixed with antioxidant formulation or theantioxidant formulation can be applied as an overlay over the tissue sothe chemical to be tested is first in contact with the antioxidant (liketear film in the eye) and then contacts the tissue. In some cases, theantioxidant can be premixed with the test substance, and also added asan overlay to the test cells or assay matrix. Pilot studies wereconducted with a representative TP (cyclohexanol CASRN 108-93-0), TN(2,4-pentanediol 625-69-4), and FP (triethylene glycol 112-27-6)associated with ROS generation. Evaluated antioxidants included ascorbicacid (CASRN 50-81-7), L-tyrosine (CASRN 60-18-4), uric acid (CASRN69-93-2), L-cysteine (CASRN 52-90-4), and glutathione (CASRN 70-18-8).

Formulation Studies

After screening of antioxidants, a final formulation was developed andused to test a broader range of chemicals (Table 3). These included fourmispredicted FPs associated with ROS; [2-(2-ethoxyethoxy) ethanol CASRN11-90-0, triethylene glycol CASRN 112-27-6, ethylene glycol diethylether CASRN 629-14-1, and styrene CASRN 100-42-5] and two mispredictedFPs associated with crosslinking activity (1,9-decadiene CASRN 1647-16-1and 2-ethoxyethyl methacrylate CASRN 2370-63-0). Controls includedcyclohexanol CASRN 108-93-0 (positive control), 2,4-pentanediol CASRN625-69-4, and dodecane CASRN 112-40-3 (negative controls), and chemicalspreviously classified as FPs that were not identified as generating ROSor having CL chemistries (triphenyl phosphite CASRN 101-02-0, ethylacetate CASRN 141-78-6, 2,4-pentanedione CASRN 123-54-6, and2,2-dimethyl-3-pentanol CASRN 3970-62-5).

TABLE 3 Literature review of in vitro eye test chemicals includingcommon false positives Chemical Catalog in vivo Name CASRN #^(b) GHS^(a)OptiSafe ROS/CL Description Reference Cyclohexanol 108- 105899 1 TP NoNo evidence Fisher, 2000. 93-0 for ROS/CL Frater, 2009. 2,4-Pentanediol625- 156019 NC TN No No evidence Tei, 2002. 69-4 for ROS/CL Wang, 2003.Dodecane 112- 297879 NC TN No No evidence Chen, 2019. 40-3 for ROS/CLHuber, 2004. 2-(2- 111- 537616 NC FP ROS Forms Adedara, Ethoxyethoxy)90-0 hydrogen 2014. Bodin, ethanol peroxide and 2003. hydroperoxidesTriethylene 112- 95126 NC FP ROS Forms Zhu, 2012. glycol 27-6 hydrogenMikulas, peroxide and 2018. hydroperoxides Ethylene glycol 629- 224111NC FP ROS Forms Di diethyl ether 14-1 hydrogen Tommaso, peroxide and2011. Clark, hydroperoxides 2001. Styrene 100- S4972 NC FP ROS Formsstyrene Zhang, 2017. 42-5 oxide Belvedere, 1981. Carlson, 2006. Niaz,2017 1,9-Decadiene 1647- 118303 NC FP CL Used as a Palmlof, 16-1crosslinker to 2000. promote Smedberg, polymerization 1997.2-Ethoxyethyl 2370- 280666 NC FP CL Used as a Chirila, 1991.methacrylate 63-0 crosslinker to Garcia, promote 2002. polymerizationFaraguna, 2015. Triphenyl 101- T84654 NC FP No No evidence Schwetlick,phosphite 02-0 for ROS/CL 1987, 1995. Kovacs, 1973. Liu, 2019. Ethylacetate 141- 270989 NC FP No No evidence Suksomtip, 78-6 for ROS/CL2010. Nakchat, 2014. 2,4- 123- P7754 NC FP No No evidence Mottley,Pentanedione 54-6 for ROS/CL 1991. Rodrigues, 2006. 2,2-Dimethyl-3-3970- D17362 NC FP No No evidence Dwarakanath, 1998. Gierke, 1999.pentanol 62-5 2 for ROS/CL Hurley, 2008. CASRN = Chemical AbstractsService Registry Number; GHS = Globally Harmonized System ofclassification and labelling of chemicals; NC = Not classified; ROS =Reactive oxygen species; CL = Crosslinker. ^(a)Lebrun et al., 2020.

Results

A limited set of chemicals were used to screen for the effects of tearantioxidants on FPs using approximate in vivo concentrations. FIG. 1shows results for this study, which included a positive control(cyclohexanol) that remained far above the OPTISAFE (now called OS2)cutoff for GHS category 1 classification. Likewise, the negative control(2,4 pentanediol) remained below the cutoff for GHS NC classification.By. contrast, the FP, triethylene glycol, showed a significant reductionin the OPTISAFE (OS2) score in response to the addition of ascorbic acid(567 μM) resulting in a change of classification from a FP to a TN.

Based on the success of the screening study, a final formulation ofantioxidant additives to the OPTISAFE (now called OS2) test kit wasdeveloped, and the effects on the OPTISAFE scores were furtherinvestigated with titrations and a broader range of FPs and controls.The exact formulation of the OPTISAFE assay is disclosed in U.S. Pat.No. 10,041,922B2; incorporated herein in its entirety. A set ofmaterials associated with ROS, CL, “other FPs” (no association with ROSor CL identified), and positive and negative controls were selected toevaluate the final formulation. Results are shown in FIG. 2 . Note thatthere is a specific reduction in the OPTISAFE (OS2) score at the in vivoconcentration for each of the chemicals with associated ROS or CLactivity. On the other hand, there was no significant change in controlsor FP not associated with ROS or CL.

For OPTISAFE (OS2), the effective concentrations showing false positivereduction with the addition of ascorbic acid into the formulation beginat 0.270 mM and the maximal effect is shown at the in vivo concentrationof 0.530 mM. This reduction of previously over-predicted chemicals wastested to be stable up to a concentration of at least 8.480 mM. Thetested concentration range of ascorbic acid showing effectiveness inreducing false positives for in vitro assays was 0.27 mM to 17 mM. Wepredict, based on the data, that any ascorbic acid concentration withinthe range of 0.27 mM and 60 mM would be effective in reducing falsepositives; indeed, as long as the ascorbic acid is soluble andeffectively buffered, there is no upper concentration limit.

Another set of experiments compared triplicate repeats for theformulation without and with antioxidants at the in vivo concentration.Table 4 shows these results for controls and Table 5 shows these resultsfor ROS and CL chemicals. Controls were unchanged.

TABLE 4 OPTISAFE (original formulation without antioxidants) and OS2(new formulation with antioxidants) Score Comparison of ControlAntioxidant Titrations OPTISAFE OS2 Score Chemical Name GHS ConditionScore AVG AVG Cyclohexanol (108-93-0) Cat. 1 Pos. 52.6 ± 1.1  66.9 ±4.9  2,4-Pentanediol NC Neg. 12.0 ± 1.4  10.2 ± 0.7  (625-69-4) Dodecane(112-40-3) NC Neg. 0.9 ± 0.8 3.0 ± 2.6 Triphenyl phosphite NC AO 113.3 ±2.1  169.0 ± 9.9  (101-02-0) Ethyl acetate (141-78-6) NC AO 23.9 ± 2.0 25.6 ± 2.4  2,4-Pentanedione NC NOT 45.3 ± 1.4  42.3 ± 6.6  (123-54-6)ROS/CL 2,2-Dimethyl-3-pentanol NC NOT 19.2 ± 1.2  15.1 ± 0.4 (3970-62-5) ROS/CL Scores of triplicate assays for FIG. 2 Controlantioxidant titrations. OS2 = OPTISAFE2, new and optimized version ofOPTISAFE; GHS = Globally Harmonized System of classification andlabelling of chemicals; Cat. = Category; NC = Not classified; Pos. =Positive control; Neg. = Negative control; AO = Antioxidant; ROS =Reactive oxygen species; CL = Crosslinker; AVG = Average

TABLE 5 OPTISAFE and OS2 Score Comparison of ROS/CL AntioxidantTitrations GHS OPTISAFE OS2 (animal (no antioxidant) (with antioxidant)Chemical Name response) Chemistry Score AVG Score AVG2-(2-Ethoxyethoxy)ethanol NC ROS 21.7 ± 0.3  12.0 ± 0.3  (111-90-0)Triethylene glycol (112-27-6) NC ROS 13.2 ± 3.5  5.1 ± 1.7 Ethyleneglycol diethyl ether NC ROS 25.4 ± 1.8  21.6 ± 2.5  (629-14-1) Styrene(100-42-5) NC ROS 53.5 ± 10.1 3.5 ± 0.8 1,9-Decadiene (1647-16-1) NC CL22.5 ± 3.6  11.4 ± 6.4  2-Ethoxyethyl methacrylate NC CL 97.1 ± 18.214.5 ± 2.7  (2370-63-0) Scores of triplicate assays for FIG. 3 ROS/CL.OS2 = OPTISAFE2, OPTISAFE with antioxidants; GHS = Globally HarmonizedSystem of classification and labelling of chemicals; NC = Notclassified; AO = Antioxidant; ROS = Reactive oxygen species; CL =Crosslinker; AVG = Average

On the other hand, all of the chemicals associated with ROS or CL hadsome reduction in score and most had an improvement in classification.As an example of this, we show for the first time that the addition ofknown antioxidants present in tears to the OPTISAFE (now called OS2)formulation reduces the OPTISAFE irritation score of FP chemicals thathave oxidative or reactive chemistries. This finding suggests that amajor drawback of many if not all in vivo alternative irritation testsis the failure to account for the effect of tears on modifying theirritation potential of test chemicals, particularly when they containoxidative and reactive chemistries.

Results demonstrate that antioxidants specifically reduce the FP ratesof chemicals associated with ROS and CL.

In order to demonstrate the effects of specific antioxidants and notincrease the FN rate, a large number of chemicals from prior OPTISAFE(original formulation without antioxidants) validation studies wereretested with the new OPTISAFE (OS2, formulation with antioxidants)formulation that includes antioxidants.

Next a study was conducted to determine if the addition of antioxidantschanges the reliability (repeatability) of the test method. After this,a large study determined if antioxidants specifically reduce the falsepositive rate without impacting the true positive rate and the truenegative rate and then all prior test chemicals were rested withantioxidants in triplicate. The OPTISAFE method was conducted aspreviously described (Choksi et al., 2020). Briefly, samples wereinitially evaluated for solubility, pH, and foaming using a standardizedprocedure. Based on the outcomes, a decision tree was followed to allowfor standardized procedural modifications for substances with thefollowing properties: 1) extreme pH, 2) insolubility, and 3) categorizedas a surfactant.

Previous studies evaluated a range of antioxidants and enzymesassociated with ocular antioxidant capabilities. The intralaboratoryrepeatability results are shown in Table 6. Three chemicals (1-octanolCASRN 111-87-5, 2,4-pentanediol CASRN 625-69-4, and triethylene glycolCASRN 112-27-6) were selected to assess the lot-to-lot repeatability ofthe new formulation with antioxidants. The three chemicals arerepresentative of a negative control, a positive control, and anOPTISAFE (original formulation without antioxidants) overpredicted invivo negative. The 10 independent repeats using different lots indicatethe variability of both the classifications and scores. For the positivecontrol, 1-octanol CASRN 111-87-5, scores ranged from 16.5 to 22.8 withan average of 19.1 and a standard deviation of 1.94. The negativecontrol, 2,4-pentanediol CASRN 625-69-4, ranged from 7.8 to 10.9 with anaverage of 9.5 and standard deviation of 0.97. The scores of triethyleneglycol CASRN 112-27-6, a previous FP identified as a potential ROSgenerator ranged from 2.1 to 4.2 with an average of 3.1 and a standarddeviation of 0.64. These results indicate that the addition ofantioxidants does not change the reliability of the test and the changefrom a FP to TN (see TG result) is consistent between numerous (10 lots)production lots and the effect is repeatable with low variability(standard deviation=0.64).

TABLE 6 Intralaboratory Repeatability of OS2 (new formulation withantioxidants) Lot # OCT PENT TEG 6.049975 22.8 10.1 3.9 6.049973 22.310.2 3.5 6.049971 18.6 10.1 3.1 6.049969 19.5 10.1 2.7 6.049967 19.9 8.04.2 6.049965 18.0 10.9 3.1 6.049963 17.8 9.8 3.4 6.049961 16.5 7.8 2.56.049959 17.6 9.2 2.4 6.049957 18.3 8.8 2.1 Score 19.1 9.5 3.1 Std Dev1.94 0.97 0.64 OCT = 1-Octanol (CASRN 111-87-5), in vivo GHS Category 2;PENT = 2,4-Pentanediol (CASRN 625-69-4), in vivo GHS NC; TEG =Triethylene glycol (CASRN 112-27-6), in vivo GHS NC; NC = Notclassified; CASRN = Chemical Abstracts Service Registry Number; GHS =Globally Harmonized System of classification and labeling of chemicals;Score = Average score; Std Dev = Standard deviation.

In addition, the repeatability for a large number of tested chemicalswas similar or better than the formulation without antioxidants. Theintralaboratory repeatability of OS2 (new formulation with antioxidants)for the different studies ranged from 95.5% to 100% with a totalrepeatability of 96.7%. Comparatively, the intralaboratory repeatabilityof OPTISAFE (original formulation without antioxidants) was 93% to 99%for the coded transferability phase (Choksi et al., 2020). Theintralaboratory repeatability OS2 (new formulation with antioxidants)for all results combined was 96.7%. Of the 426 assays run using OS2 (newformulation with antioxidants), 412 of the repeats were in agreement.This data demonstrates that for 10 different lots of the new formulationthe test method is repeatable, and antioxidants do not change thereliability of the test method.

Next, to demonstrate that the addition of antioxidants only improves thefalse positive rate but does not change the TP or TN rates, a majorstudy tested ALL prior OptiSafe validation chemicals in triplicate (OS2)and compared to the prior validation studies without antioxidant results(OS). These results are shown in Table 7.

Tables 7A and 7B Consensus OptiSafe Results Comparing OriginalFormulation (without Antioxidants) and OS2 (New Formulation withAntioxidants)

TABLE 7A Past Transferability Study Classifications and TriplicateRepeats OptiSafe Past Transferability Study Classifications andTriplicate Repeats by OS2 In vivo OS OS2 OS2 OS2 OS2 n Chemical Name(CASRN) GHS Cons¹ R1 R2 R3 Cons 1 1,3-Di-iso-propylbenzene (99-62-7) NC1 2/1 2/1 2/1 2/1 2 n-Hexyl bromide (111-25-1) NC NC NC NC NC NC 3iso-Octyl acrylate (29590-42-9) NC NC NC NC NC NC 4 Glycerol (56-81-5)NC NC NC NC NC NC 5 1,9-Decadiene (1647-16-1) NC 2 NC 2 NC NC 6Di-iso-butyl ketone (108-83-8) NC NC NC NC NC NC 71-Bromo-4-chlorobutane (6940-78-9) NC NC NC NC NC NC 8 1,6-Dibromohexane(629-03-8) NC NC NC NC NC NC 9 n-Octyl bromide (111-83-1) NC NC NC NC NCNC 10 Propylene glycol (57-55-6) NC NC NC NC NC NC 11 2,4-Pentanediol(625-69-4) NC NC NC NC NC NC 12 Potassium tetrafluoroborate (14075-53-7)NC NC NC NC NC NC 13 4,4-Methylene bis-(2,6-ditert-butyl)phenol NC CNMCNM CNM CNM CNM (118-82-1) 14 2,2-Dimethyl-3-pentanol (3970-62-5) NC 2NC 2 NC NC 15 2-Methyl-1-pentanol (105-30-6) 2B 2 2 2 2 2 16 Sodiumchloroacetate (3926-62-3) 2B 1 1 1 1 1 17 Isobutyraldehyde (78-84-2) 2B1 1 1 1 1 18 Camphene (79-92-5) 2B 2 2 2 2 2 19 Ammonium nitrate(6484-52-2) Cat 2A 2 2 2 2 2 20 3,3-Dithiodipropionic acid (1119-62-6)2B 1 1 1 1 1 21 Isobutanol (78-83-1) 2A 1 1 1 1 1 22 Dibenzyl phosphate(1623-08-1) 2A 1 1 1 1 1 23 Propasol solvent P (1569-01-3) 2A 2 2 2 2 224 Methyl cyanoacetate (105-34-0) 2A 1 1 1 1 1 25 n-Butanol (71-36-3)1/2A 1 1 1 1 1 26 3,4-Dichlorophenyl isocyanate (102-36-3) 1 1 1 1 1 127 p-Tert-butylphenol (98-54-4) 1 1 1 1 1 1 The OptiSafe (originalformulation without antioxidants) consensus result from the pasttransferability study (Choksi et al 2020), OS2 (new formulation withantioxidants) triplicate repeat data, and OS2 consensus data for thesame chemical set is shown. In vivo GHS = GHS classifications based onthe Draize data (the reference values); categories NC, 2A/B, 1 and IVare the decreasing irritancy categories determined through this system.R1, R2, R3, and Cons = repeats 1, 2, and 3, and consensus of therepeats. CNM = ″criteria not met″ due to internal quality assurance.OptiSafe and OS2 consensus are the majority prediction based on thetriplicate assays conducted for each test method. OS = OptiSafe; CASRN =Chemical Abstracts Service Registry Number; GHS = Globally HarmonizedSystem of classification and labeling of chemicals; NC = not classified;OS2 = OptiSafe2, new and optimized version of OptiSafe. ¹Choksi et al.,2020.

TABLE 7B Past OptiSafe (original formulation without antioxidants)Application Domain Study Classifications and Triplicate Repeats by OS2(new formulation with antioxidants) Past OptiSafe Application DomainStudy Classifications and Triplicate Repeats by OS2 In vivo OS OS2 OS2OS2 OS2 n Chemical Name (CASRN) GHS Cons¹ R1 R2 R3 Cons 28Cyclopentasiloxane (541-02-6) NC NC NC NC NC NC 29 Ethylene glycoldiethyl ether (629-14-1) NC 2 2 2 2 2 30 Hexane (110-54-3) NC NC NC NCNC NC 31 2-Ethylhexylthioglycolate (7659-86-1) NC NC NC NC NC NC 32iso-Propyl bromide (75-26-3) NC NC NC NC NC NC 33 1,2,6-Hexanetriol(106-69-4) NC NC NC NC NC NC 34 3-Methoxy-1,2-propanediol (623-39-2) NCNC NC NC NC NC 35 Triethylene glycol (112-27-6) NC 2 NC NC NC NC 36*Triphenyl phosphite (101 -02-0) NC 1 1 1 1 1 37 2-Ethoxyethylmethacrylate (2370-63-0) NC 1 2 2 NC 2 38 Hexamethyldisiloxane(107-46-0) NC NC NC NC NC NC 39 Hexyl cinnamic aldehyde (101-86-0) NC NCNC NC NC NC 40 p-Methyl thiobenzaldehyde (3446-89-7) NC 1 1 1 1 1 41Triclocarban (101-20-2) NC CNM CNM CNM CNM CNM 42 Ethyl acetate(141-78-6) NC 2 2 2 2 2 43 2,4-Pentanedione (123-54-6) NC 2 2 1 2 2 44Dodecane (112-40-3) NC NC NC NC NC NC 45 2-(2-Ethoxyethoxy)ethanol(111-90-0) NC 2 NC NC NC NC 46 n,n-Dimethylguanidine sulfate (598-65-2)NC 2 NC NC NC NC 47 1,4-Dibromobutane (110-52-1) NC NC NC NC NC NC 483-Phenoxybenzyl alcohol (13826-35-2) NC 2 2 1 2 2 49 Styrene (100-42-5)NC 1 NC NC NC NC 50 1,5-Hexadiene (592-42-7) NC CNM NC NC NC NC 51n,n-Diethyl-m-toluamide (134-62-3) 2B 2 1 1 1 1 52 3-Chloropropionitrile(542-76-7) 2B 1 1 1 1 1 53 Isopropyl acetoacetate (542-08-5) 2B 1 1 1 11 54 n-Butanal (123-72-8) 2B 1 1 1 1 1 55 Ethyl-2-methyl acetoacetate(609-14-3) 2B 1 1 1 1 1 56 Maneb (solid) (12427-38-2) 2B 1 CNM CNM CNMCNM 57 Isopropanol (67-63-0) 2A 2 2 2 2 2 58 2-Amino-3-pyridinol(16867-03-1) 2A CNM CNM CNM CNM CNM 59 Allyl alcohol (107-18-6) 2A 1 1 11 1 60 Xylene (1330-20-7) NC NC NC NC NC NC 61 Cyclopentanol (96-41-3)2A 1 1 1 1 1 62 n-Hexanol (111-27-3) 2A 2 2 2 1 2 63 gamma-Butyrolactone(96-48-0) 2A 2 2 2 2 2 64 n-Octanol (111-87-5) 2A 2 2 2 2 2 65 Methylacetate (79-20-9) 2A 2 2 2 2 2 66 2,6-Dichlorobenzoyl chloride(4659-45-4) 2A 1 1 1 1 1 67 Acetone (67-64-1) 2A 2 2 2 2 2 68Methylthioglycolate (2365-48-2) 1 1 1 1 1 1 69 Diethylaminopropionitrile(5351 -04-2) 1 CNM 2 1 1 1 70 6-Methyl purine (2004-03-7) 2B 2 or 1 2/12/1 1 2/1 71 Imidazole (288-32-4) 1 CNM 1 1 1 1 72 Sodium perboratetetrahydrate (10486-00-7) 1 CNM 1 1 1 1 73 2,5-Dimethylhexanediol(110-03-2) 1 2 2 1 1 1 74 Butanedioic acid, sulfo-,1,4-bis(2-ethylhexyl) 1 2 2 2 2 2 ester, sodium salt (577-11-7) 75Cyclohexanol (108-93-0) 1 1 1 1 1 1 76 Lactic Acid (50-21-5) 1 1 1 1 1 177 Protectol PP (80-54-6) 1 1 1 1 1 1 78 Lauric acid (143-07-7) 1 1 1 11 1 The OptiSafe (original formulation without antioxidants) consensusresult from the past application domain study (Choksi et al 2020), OS2(new formulation with antioxidants) triplicate repeat data, and OS2consensus data for the same chemical set is shown. In vivo GHS = GHSclassifications based on the Draize data (the reference values);categories NC, 2A/B, 1 and IV are the decreasing irritancy categoriesdetermined through this system. R1, R2, R3, and Cons = repeats 1, 2, and3, and consensus of the repeats. CNM = ″criteria not met″ due tointernal quality assurance. OptiSafe and OS2 consensus are the majorityprediction based on the triplicate assays conducted for each testmethod. *Triphenyl phosphite CASRN 101-02-0, five total repeats wereconducted for an overall prediction of GHS category 1. Only three of therepeats are shown. OS = OptiSafe; CASRN = Chemical Abstracts ServiceRegistry Number; GHS = Globally Harmonized System of classification andlabeling of chemicals; NC = not classified; OS2 = OptiSafe2, new andoptimized version of OptiSafe. ¹Choksi et al., 2020.

Table 8 shows a comparison of the accuracy (for the detection of GHS NC)of OPTISAFE (original formulation without antioxidants) and OS2 (newformulation with antioxidants). Based on the in-house retesting ofchemicals from the prior validation study (transferability andapplication domain) for OS2 (new formulation with antioxidants), the FPrate for the GHS NC prediction improved from 40.0% for OPTISAFE(original formulation without antioxidants) to 22.2% for OS2 (newformulation with antioxidants). The FN rate for both remained the sameat 0.0%, and the overall accuracy improved from 80.3% for OPTISAFE(original formulation without antioxidants) to 89.2% for OS2 (newformulation with antioxidants). This demonstrates the effect ofantioxidants is specific to the reduction of the false positive (FN)rate; the false negative (FN) rate remained at 0 and accuracy improvedfrom 80% to almost 90%.

TABLE 8 OptiSafe (original formulation without antioxidants) CodedValidation Study Results Compared with Repeat Testing by OS2 (newformulation with antioxidants) Statistics ^(1,2)OptiSafe OS2 FN Rate 0.0% (0/36)  0.0% (0/38) FP Rate 40.0% (14/35) 22.2% (8/36) Accuracy80.3% (57/71) 89.2% (66/74) OS = OptiSafe; OS2 = OptiSafe2; FN = falsenegative; FP = false positive. The values in parentheses denote thenumber of tested chemicals within each statistic over the total numberof chemicals. The total n for OptiSafe and the n for OS2 do not matchdue to chemicals not meeting criteria of each test method. A fullside-by-side review of the n's for OS and OS2 can be found in Table 3.¹Choksi et al., 2020. ²Lebrun et al., 2019.

The FN rate, FP rate, and accuracy for just the 12 surfactants was 0%(0/5), 16.7% (1/6), and 90.9% (10/11), respectively. Sodium lauroylsarcosinate (10%) did not meet criteria (CNM) due to assay inhibition(see Choksi et al 2020). As shown in Table 9A, considering all results(including surfactants) for the prediction of the GHS NC classification,the FN rate was 0% (0/86), the FP rate was 20.8% (10/48), and theaccuracy was 92.5% (124/134).

TABLE 9A OS2 (new formulation with antioxidants) Repeat Testing of AllOptiSafe (original formulation without antioxidants) Validation Studiesand Additional Surfactants for the Detection of GHS NC Statistics OS2 NCFN Rate  0.0% (0/86) FP Rate 20.8% (10/48) Accuracy 92.5% (124/134) GHSNC vs. 2, 1 analysis. Analysis includes all complied OS2 data (repeatsof OptiSafe transferability study, repeats of OptiSafe applicationdomain study, repeats of OptiSafe retrospective study, repeats ofOptiSafe expanded corrosive study, and expanded surfactant study). Thevalues in parentheses denote the number of indicated chemicals withineach statistic over the total number of chemicals. OS2 = OptiSafe2, newand optimized version of OptiSafe; NC = not classified; FN = falsenegative; FP = false positive.

For the detection of ocular corrosives (GHS category 1), there were atotal of 122 triplicate results. This is because 12 results predicted aseither Category 2 or 1 (no differentiation between irritant andcorrosive due to objective internal criteria) are not included in thisanalysis. As shown in Table 9B, the FN rate was 10.6% (5/47), with noGHS category 1 chemicals mispredicted as NC (all category 1 FNs werepredicted as GHS category 2). The FP rate was 26.7% (20/75), and theaccuracy was 79.5% (97/122).

TABLE 9B OS2 (new formulation with antioxidants) Repeat Testing of AllOptiSafe (original formulation without antioxidants) Validation Studiesand Additional Surfactants for the Detection of GHS Category 1Statistics OS2 Cat 1 FN Rate 10.6% (5/47) FP Rate 26.7% (20/75) Accuracy79.5% (97/122) GHS Cat 1 vs. 2, NC analysis. Analysis includes allcomplied OS2 data (repeats of OptiSafe transferability study, repeats ofOptiSafe application domain study, repeats of OptiSafe retrospectivestudy, repeats of OptiSafe expanded corrosive study, and expandedsurfactant study). The values in parentheses denote the number ofindicated chemicals within each statistic over the total number ofchemicals. OS2 = OptiSafe2, new and optimized version of OptiSafe; NC =not classified; FN = false negative; FP = false positive.Comparison of OS2 (with Antioxidants) to Other In Vitro OcularIrritation Tests

The performance of OS2 (new formulation with antioxidants) was comparedto other test methods (without antioxidants) using OECD guidelinestatistics for the lowest (GHS NC) and highest (GHS Category 1)classification. For the GHS NC versus the rest comparison, OS2 (newformulation with antioxidants) has an accuracy of 92.5%, a falsenegative rate (FNR) of 0.0%, and a false positive rate (FPR) of 20.8%(Table 10A). For the GHS Category 1 versus the rest comparison, OS2 (newformulation with antioxidants) has an accuracy of 79.5%, a FNR of 10.6%,and a FPR of 26.7% (Table 10B).

Table 10. Comparison of OS2 (New Formulation with Antioxidants) withOECD Published Accuracies for BCOP (OPKIT), BCOP (LLBO), EPIOCULAR, ICE,OCULAR IRRITECTION and STE

TABLE 10A GHS NC vs. Rest OS2 BCOP (OPKIT)¹ BCOP (LLBO)¹ Epi² ICE³ FNR =0.0% (0/86) FNR = 0.0% (0/107) FNR = 6.3% (6.5/104) FNR = 4.2% (2.4/57)FNR = 3.0% (3/101) FPR = 20.8% (10/48) FPR = 68.5% (61/89) FPR = 45.1%(18.5/41) FPR = 36.9% (20.3/55) FPR = 24.1% (20/83) Acc. = 92.5%(124/134) Acc. = 68.9% (135/196) Acc. = 82.8% (120/145) Acc. = 79.6%(89.2/112) Acc. = 87.5% (161/184) Bal. Acc. = 89.6% Bal. Acc. = 65.8%Bal. Acc. = 74.3% Bal. Acc. = 79.5% Bal. Acc. = 86.5% OI⁴ STE⁵ FNR =9.3% (4.3/46) FNR = 12.3% (9/73) FPR = 41.2% (17.7/43) FPR = 19.3%(11/57) Acc. = 75.3% (67.0/89) Acc. = 8.6% (110/130) Bal. Acc. = 74.8%Bal. Acc. = 84.2%

TABLE 10B GHS Cat. 1 vs. Rest OS2 BCOP (OPKIT)¹ BCOP (LLBO)¹ ICE³ OI⁴STE⁵ FNR = 10.6% (5/47) FNR = 13.8% (9/65) FNR = 24.1% (13.5/56) FNR =46.7% (21/45) FNR = 46.5% (9.3/20) FNR = 51.3% (20/39) FPR = 26.7%(20/75) FPR = 25.4% (32/126) FPR = 20.8% (18.5/89) FPR = 7.1% (9/127)FPR = 19.1% (13.2/69) FPR = 1.2% (1/86) Acc. = 79.5% Acc. = 78.5% Acc. =77.9% Acc. = 82.6% Acc. = 74.7% Acc. = 83.2% (97/122) (150/191)(113/145) (142/172) (66.5/89) (104/125) Bal. Acc. = 81.4% Bal. Acc. =80.4% Bal. Acc. = 77.6% Bal. Acc. = 73.1% Bal. Acc. = 67.2% Bal. Acc. =73.8%Comparison of OS2 (new formulation with antioxidants) with other oculartest methods. Table 10A compares the statistics for classification ofGHS NC versus Cat. 2 or 1. Table 10B compares the statistics of GHS Cat.1 versus NC or Cat. 2. EpiOcular is unable to detect Cat. 1 chemicalsand therefore is not listed on Table 7B (OECD, 2019a). OS2=OptiSafe2,new and optimized version of OptiSafe; GHS=Globally Harmonized System ofclassification and labeling of chemicals; BCOP=Bovine Corneal Opacityand Permeability; LLBO=Laser light-based opacitometer; Epi=EpiOcular;ICE=Isolated Chicken Eye; OI=Ocular Irritection; STE=Short TimeExposure; OECD=Organization for Economic Co-operation and Development;Cat.=Category; FNR=False negative rate; FPR=False positive rate; NC=Notclassified; Acc.=Accuracy; Bal. Acc.=Balanced accuracy.

-   -   ¹OECD, 2020a    -   ²OECD, 2019a    -   ³ECD, 2018    -   ⁴ECD, 2019b    -   ⁵OECD, 2020b

Table 10A and Table 10B compares OS2 (new formulation with antioxidants)with the stated OECD guideline accuracies for both the detection of GHSNC and GHS Category 1 or other tests (all without antioxidants).Balanced accuracy (Bal. Acc.) is included because the numbers ofnegatives and positives (shown in parenthesis) are variable and balancedaccuracy provides an accuracy that accounts for true positives and truenegatives equally. For the detection of NC versus the rest, the additionof oxidants significantly reduced the FPR as compared to the other testsexcept for the STE methods. However, the STE method has a much higherFNR. Reducing the FPR while maintaining a low FNR is an importantconsideration. Unless the FPR is low, it becomes unclear if positivesare true positives or false positives, and this puts resistance toadopting nonanimal tests because safe products are erroneouslyclassified as unsafe for the eye.

Previously, we demonstrated that the best way to compare one test toanother is by comparing results for the same chemical (Lebrun et al.,2020). The comparison of OS2 (new formulation with antioxidants) withother test methods for the detection of GHS NC is shown in Table 11.When performance for the same chemicals is evaluated, OS2 (newformulation with antioxidants) has a comparatively higher accuracy thanthe other tests to which it was compared (without antioxidants) ofaround 90%. The FPR for the same chemicals is about ⅓ to ½ of comparedto the other tests. This is consistent with the overall accuracy thatresulted from the 131 results in triplicate. Also noteworthy, is thefinding that the OS2 (new formulation with antioxidants) has a falsenegative rate of 0.0% (indicating the addition of antioxidants does notresult in false negatives), compared to the other tests which have falsenegatives.

TABLE 11 Accuracy Comparison for the Same Chemicals between OS2 (newformulation with antioxidants) and BCOP (LLBO), BCOP (OP-KIT),EPIOCULAR, ICE, OCULAR IRRITECTION, STE Comparison FNR FPR Accuracy OS2 0.0 (0/54) 20.0 (3/15) 95.7 (66/69) BCOP (LLBO)  5.6 (3/54) 33.3 (5/15)88.4 (61/69) OS2  0.0 (0/65) 30.8 (4/13) 94.9 (74/78) BCOP (OP-KIT)  7.7(5/65) 38.5 (5/13) 87.2 (68/78) OS2  0.0 (0/66) 16.0 (4/25) 95.6 (87/91)EpiOcular  1.5 (1/66) 32.0 (8/25) 90.1 (82/91) OS2  0.0 (0/32) 42.9(3/7) 92.3 (36/39) ICE  3.1 (1/32) 85.7 (6/7) 82.1 (32/39) OS2  0.0(0/30) 22.2 (6/27) 89.5 (51/57) OI  6.7 (2/30) 44.4 (12/27) 75.4 (43/57)OS2  0.0 (0/45)  9.7 (3/31) 96.1 (73/76) STE 24.4 (11/45)  9.7 (3/31)81.6 (62/76) Comparison of same chemicals between OS2 (new formulationwith antioxidants) and other ocular test methods. n = number ofchemicals in common between the test methods compared; OS2 = OptiSafe2;new and optimized version of OptiSafe; BCOP = Bovine Corneal Opacity andPermeability; LLBO = Laser light-based opacitometer; ICE = IsolatedChicken Eye; OI = Ocular Irritection; STE = Short Time Exposure; FNR =False negative rate, expressed as %, number of positives misclassifiedas negatives[((FN)/(FN + TP)) · 100]; FPR = False positive rate,expressed as %, number of negatives misclassified as positives[((FP)/(FP + TN)) · 100]; Accuracy = total correct predictions dividedby the n in common. [((TP + TN)/(TP + TN + FP + FN)) · 100]; TP = TruePositive; TN = True Negative. Chemical results obtained from Lebrun etal., 2020; Lebrun et al., 2021 for same chemical comparisons between OS2and the test methods.

Based on the dramatic reduction of the false-positive rate (from about40% to 20%), without any increase in the false negative rate, weconcluded that the addition of antioxidants to nonanimal tests iscritical to lower the false-positive rate and have a high accuracy. Thisdramatic improvement is unexpected, and while nonanimal tests for eyesafety have been done for 25 years, only now has the importance of theaddition of antioxidants to these tests been recognized. The addition ofantioxidants appears to be required for the accurate and specificmodeling of eye safety after chemical or product exposure. This hasnever before been described with respect to in vitro, nonanimal testmethods.

Ascorbic acid is a water-soluble essential nutrient and is more highlyconcentrated in the tear film than in the serum. Its main functions areas an electron donor/antioxidant and cofactor for certain dioxygenasesin epigenetic regulation (Han et al., 2021). Other roles in the humanbody include cell-signaling, as a hormone growth factor, and cytokine,including possibly via sodium-dependent vitamin C transporter 2 (SVCT)mediation of Janus kinase 2 (JAK), which s promotes regulation ofvitamin C in epigenetic modifications, and complex effects related tothe regulation of cell pluripotency and differentiation (Han et al.,2021). Other functions include immune system modulation (Carr andMaggini, 2017).

Ascorbic acid induces collagen secretion and formation of cell sheets inthe eye (human corneal cell culture; Grobe and Reichl, 2013). Ascorbicacid is a required cofactor for the hydroxylation of the amino acidsproline and lysine required for collagen triple helix formation andstabilization, including in tissue repair (Levene and Bates, 1975; Grobeand Reichl, 2013; Peterkofsky, 1972). Collagen is critical tomaintaining eye health and function; including the stroma (collagen I)and the basement membrane (collagen IV).

Because cell and excised eye assays could respond to ascorbic acid byaltering cell growth, repair or other metabolic responses or possibly asa “nutritional response” (impacting the collagens etc.), as discussedabove, it has been unclear (variables left undefined) if the ascorbicacid in tear, or added ascorbic acid, specifically inactivates reactivemolecules and this prevents damage from occurring in the first place(versus the other types of responses mentioned above). On the otherhand, the cell free macromolecular test system that we used specificallymeasures the level of molecular damage. By using a cell free testsystem, one can rule out effects on cell growth and repair, nutritionand possibly other complex yet to be defined variables related to cellsand tissues; and more specifically determine if the mechanism of actionis specific to the inactivation of ROS by direct chemical reduction(provides an electron to stabilize ROS). Therefore, the antioxidantprovides immediate protection against ROS and other toxins. The responseof a complex biological system is not required. However, becauseantioxidants such as ascorbic acid can buffer to extreme pH, and extremepH in itself is damaging to the eye, these must be highly buffered (tobetween pH 6.5-7.5) before coming into contact with the eye. In ourexperience, HEPES, Tris and bicarbonate (for CO2 systems) are alleffective buffers. In addition, the addition of dextran and albumin willimprove the viscosity, retention time on the eye and osmolarity,ensuring that the antioxidant solution stays on the eye but does not dryout the eye causing additional damage while it remains (sticks) on theeye increasing the duration that the solution can interact (inactivate)toxin on the surface or that has penetrated into the tissue.

This formulation would be particularly effective after an accidentalspill or other exposure to a strong oxidizer, which typically penetrateinto the tissue. Likewise, in some formulations including make-up,cosmetics and personal care products (together, personal care products),preservatives and other toxins damage tissue by oxidation. Includingthis antioxidant mix in the formulation of the personal care productwill likely decrease the irritating effects of the formulation byinactivating these reactive molecules before they damage the eye. In asimilar fashion, eye drops and eye medications can contain preservativesand chemicals that increase penetration of the drug. These includebenzalkonium chloride and other preservatives and agents that allowmedications to penetrate into the tissue. By adding a buffered solutionof ascorbic acid to the formulation, the reactive oxygen species arecontrolled resulting in less tissue damage and less of an adverse oculareffect. As explained above, the reduction in adverse effects is notrelated to complex biological response, it is related to the specificand immediate quenching of reactive chemistries by antioxidant electrondonation. Per the examples provided, the specificity of the reaction canbe verified using cell free eye irritation test; in this case, thesubstance being tested is predicted as an irritant because it damagesmacromolecules in a cell free test system without the antioxidantformulation. However, when the antioxidant formulation is added, thesubstance being tested is predicted to be a nonirritant by a cell freetest system. This simple test will determine if the mechanism of actionis simple chemical inactivation prior to damaging the tissue versusother complex nutritional or hormonal effects, etc.

Antioxidant Formulations Formulations for Reducing False Positives forIn Vitro Ocular Irritation Tests

In certain embodiments of the various in vitro ocular irritation testmethods, an antioxidant formulation has been employed. That antioxidantformulation may utilize any combination of known antioxidants, and insome preferred embodiments, the formulation utilizes any one of moreantioxidants found in tears (see e.g., Table 2). More particularly,antioxidants may be selected from ascorbic acid, baicalein,beta-carotene, bilirubin, caeruloplasmin, catechin, cobalamin, coenzymeQ10, cortisone, cryptoxanthin, crystallin, curcumin, cyanidin,delphinidin, epigallocatechin-3-gallate, esculetin, estradiol, estriol,folic acid, genistein, glutathione, glutathione peroxidase, human serumalbumin, idebenone, kaempferol, L-acetylcarnitine, L-cysteine, lipoicacid, L-tyrosine, lutein, lycopene, melatonin, mexidol, myo-inositol,myricetin, N-acetyl cysteine, estrogen, omega-3, omega-6, omega-9,pelargonidin, peonidin, petunidin, piceatannol, pigment epitheliumderived factor, quercetin, resveratrol, riboflavin, selenium, silymarin,superoxide dismutase, taurine, tempol, thiamine, thioredoxin,thymoquinone, transferrin, ubiquinol-10, uric acid, vitamin A, vitaminD3, vitamin E, and zeaxanthin.

Because metals may promote oxidation and/or generation of reactiveoxygen species, the antioxidant formulation should have no metals in anyvalance state, including metal complexes (such as zinc ascorbatecomplexes). Accordingly, preferred embodiments of the formulation do notcomprise any metals, including in particular, iron, silver, magnesium,zinc, and copper.

Antioxidants, such as ascorbic acid, serve the purpose of reducingoxidative injury within biological systems through quenching of freeradicals (Gulcin, 2020). In contrast, metals cause reduction-oxidationcycling reactions which causes damaged. Through the Fenton reaction,metals, such as iron, promote oxidation and the production of freeradicals; ultimately, this results in biological injury (Winterbourn,1995). Specifically, the Fenton reaction produces hydroxyl radicals fromhydrogen peroxide and an Iron (II) catalyst. While the inclusion ofantioxidants is aimed to target this oxidative stress, there is a pointin which the “increased formation of reactive oxygen species (ROS)overwhelms body antioxidant protection and subsequently induces DNAdamage” (Jomova and Valko, 2011). These metals include iron (Fe), copper(Cu), chromium (Cr), cobalt (Co) and other metals (Jomova and Valko,2011). Furthermore, the addition of metals is counterproductive to theinitial objective. The point of interest is oxidative stress mediation;the disclosed embodiments do not align with the addition of metals, andindeed, we teach away from this.

In some embodiments, the antioxidant formulation comprises one or morecompounds selected from glutathione (about 1.0-107 μM), L-cysteine(about 0.5-5,000 μM), L-tyrosine (about 0.5-5,000 μM), ascorbic acid(about 0.27-60 mM) and uric acid (about 3.0-30,000 μM). Ascorbic acid(0.3 mg/ml and 3 mg/ml) has been used for the experiments disclosedherein.

Besides antioxidant(s), the antioxidant formulation will preferably alsoinclude serum albumin (or other serum protein). Albumin itself hasantioxidant properties, and also promotes lipophilic chemical transportand binding properties, osmotic properties, protects cells, and caninteract with toxins. The albumin concentration is between 0.05% and 10%w/v; more preferably 0.1% to 5% w/v. Although 1% w/v bovine serumalbumin was used for the experiments disclosed herein, any other speciesalbumin, and concentrations within the disclosed ranges, may be used inaccordance with the disclosed and claimed invention.

Besides antioxidants and albumin, the formulation also includes dextran.Dextran has osmotic properties, prevents drying out, improves viscosityand ensures the antioxidant/protein solution maintains a film over thetissue. Preferably, the dextran is present in a concentration of betweenabout 3% and 30% w/v, and more preferably at a concentration of at leastabout 5% w/v. Although 5% w/v dextran was used for the experimentsdisclosed herein, any other concentrations within the disclosed rangesmay be used in accordance with the disclosed and claimed invention.

The antioxidant formulation is dissolved in a salt buffer system,adjusted to a neutral pH. The buffer may be any buffer used in the art,such as HEPES, Tris or bicarbonate buffer, all of which work well inthis pH range. Sodium chloride should be added to provide osmolaritymirroring that of physiologic tears, typically normal saline; 6 mg/mlNaCl was used in these studies.

Other ingredients, including thickening agents, such as carboxymethylcellulose, moisturizer/humectant/emollient, such as glycerin, andpreservatives (although antioxidants are also useful as preservativesagainst oxidative damage), such as benzoic acid, benzyl alcohol,benzalkonium chloride, etc., may also be added, particularly if anantioxidant formulation is formulated for sale and storage at roomtemperature.

While the antioxidant formulation has been demonstrated to reduce falsepositive rate in the in vitro nonanimal ocular irritation test systems,it is also envisioned that the same formulation may be used in vivo as acountermeasure to acute exposure to eye irritants, such as mustard gas.

Formulations for Mitigating In Vivo Damage after Irritant Exposure

In certain embodiments, the antioxidant formulation developed forreducing false positives and enhancing accuracy of in vitro eyeirritation tests, can be modified for treating exposure to eyeirritants, such as a countermeasure against chemical assault or afteraccidental exposure such as a chemical splash. The results above for usein in vitro tests demonstrate that physiologic levels of tearantioxidants protect eye tissues (reconstituted and excised eye models)from oxidizers, crosslinkers and reactive oxygen species. The presenceof antioxidants (in tears) are a key difference between in vitrononanimal tests and live animals, where tears protect the live animaleye naturally allowing some chemicals to be classified as nonirritants,we hypothesized that increasing the concentration of these antioxidantsabove physiologic levels by the external application of a supplementalhigh antioxidant tear solution, will protect the eye from higherconcentrations of chemical irritants, and therefore protect against moresevere oxidative damage. Accordingly, the proposed formulations, whereantioxidant concentrations are substantially increased, are likely tohave utility for the protection from and first aid following contact ofthe eye with strong oxidizers, crosslinkers and chemicals that generatereactive oxygen species. To test this hypothesis, we added antioxidantformulations with and without high concentrations of ascorbic acid (17.0mM; 3 mg/ml; 10-fold higher than used for the in vitro studies) in areconstituted human corneal epithelium (RhCE) model system. Withreference to FIG. 4 , it can be seen that the high levels of ascorbicacid (3 mg/ml (17.0 mM) in a buffered salt solution) affordedsignificant protection against damage (cell death) caused by a varietyof damaging chemical irritants, compared to buffered salt solutionalone. The other constituents of the high concentration antioxidantformulation for in vivo post-exposure protection will be essentially thesame as the in vitro formulation, e.g., albumin and dextran, andoptionally moisturizers/humectants, thickening agents, preservatives.

Experimental Details and Examples 1.1 Additions of Eye Antioxidants toBiochemical Test

The addition of antioxidant mixtures can be done into the biochemicaltest matrix or added to sample to be tested. This is done easily throughintroduction of a specified amount to the reagent, followed bysolubilization into the reagent, for example by using a metal stir barand magnetic mixing plate for an allotted amount of time. The homogenousmixture is then used for the assay as normally conducted. One or more ofthe following antioxidants were used for final antioxidant mixture; GSH,L-cysteine, L-tyrosine, ascorbic acid, and uric acid. The procedure was:

Pretest and Surfactant Check

-   -   1) Make “10% dilution” of substance in screw cap glass test        tubes in 2 mL OSII™ Blanking Buffer (BB) by adding 200 μL/200 mg        test sample into a screw-cap glass tube.    -   2) Add 2 mL of the pH-adjusted BB to labeled tube [for colored        test samples, adjust the pH of the BB to 6.36 using dilute (0.1        N, etc.) NaOH or dilute (0.1 N, etc.) HCl].    -   3) Cap tube and invert three times. Vortex mix at 45-degree        angle at maximum speed for 10 sec.    -   4) Place tubes in a rack on bench top and allow the tube to sit        undisturbed for 5-10 min.    -   5) Inspect tube; pick up and hold to light. If all of the        substance is at the meniscus and blocks the observation, repeat        the procedure at a 1% dilution if needed. Measure from the        meniscus up using a metric ruler. If froth extends greater than        0.2 cm above the meniscus and bubbles are present (in either        tube), the substance is classified as a “surfactant” using this        method.    -   6) Inspect the 10% tube from the sides and bottom. If the        substance is a liquid and has not mixed with the OSII BB, starts        to form “oily” droplets at the top or middle or bottom, or is        unclear whether the substance is mixed (all clear liquids),        conduct the A procedure (in addition to the a procedure).

Completely Insoluble Check

-   -   1) If the substance is a solid, let the 10% solution (made        above) sit undisturbed for 30 min (up to 4 h). Note if the        substance is a solid and the majority floats to the top (“F”),        aggregates in the middle (“A”), or sinks (“S”) to the bottom of        the tube; note this and remove approximately 0.5 mL of the        liquid portion (and avoid solid portion) using a 1-mL        serological pipette. Record this in procedure notes section.    -   2) Set the spectrophotometer to 400 nm and blank.    -   3) Measure the OD400 of the recovered solution.    -   4) If the substance is a solid and the measured value is less        than 0.350, follow the 42-h completely insoluble protocol. See        Section Ci if the substance floats/aggregates/clumps or sinks.    -   5) If the OD is greater than 0.350, the substance is not        completely insoluble.

H Buffering Score Pretest

-   -   1) Measure the buffering power of an unknown and calculate the        H-buffering score by adding a 125 μL or, if solid, a 125 mg        (±10%) aliquot of test sample into a 7-mL tube.    -   2) Select the 8 mL tube of frozen Active Agent (AA). Use a 10-mL        beaker with “mini” stir bar. Prepare the small vial of the        pH-adjust solution by adding 2.5 mL deionized water and        invert/shake 5-8 times. Optional: Dilute the pH-adjust solution        1:3 and/or 1:10 in deionized water (if needed for fine pH        adjustments).    -   3) Record the starting pH of the AAII (6.36) on the pre-test        data sheet. 4) Add 1.25 mL of pH-adjusted AA into a plastic 7-mL        tube.    -   5) Cap tube and vortex mix for 5 sec at maximum speed.    -   6) Place tube on bench top and allow it to sit for 5 min±30 sec.        In cases where the solution pH does not stabilize, continue        incubation until the pH stabilizes; record this pH value and        note the time until pH stability reached.    -   7) Measure and record the final pH on the pre-test data sheet.    -   8) Subtract the final pH from the starting pH. Use this absolute        value as the exponent (base 10). Add a sub A or B to indicate        acidic or basic buffering. The resulting value is the        H-Buffering Score.    -   9) If the resulting H-Buffering Score is: Less than 5.0A or        2.0B, the substance is within the MA or Ci (floats/aggregates or        sinks) application domains; Not MA but less than 100A or 100B,        the substance is within the *HMA application domain; Greater        than 100A or B, then follow the H procedure. (*Note: In the        event a test substance is both completely insoluble and the H        score is less than 100.0 but greater than 5.0A or 2.0B, use best        scientific judgment to select one or the other.)

Alpha Procedure

-   -   1) Pre-weigh solid test samples and place membrane discs before        starting.    -   2) Adjust the pH of the BB to 6.36 using NaOH. (Only required        for colored test samples.)    -   3) Remove the 40-mL tube of frozen AAII solution from the        freezer. Record the lot number and then warm the AAII solution        in a 26-28° C. water bath. The water level should be the same        height as the AAII within the tube. The AAII should sit in the        water bath for 40 min (50 min maximum).    -   4) Transfer the AAII to a 50-mL beaker with a stir bar. Allow        good vortexing but minimal foaming. Add the antioxidant mixture        at a specified concentration. Calibrate the pH meter and confirm        its functionality by comparing measurements with a second        calibrated pH meter. Record the initial pH. Reconstitute the        larger aliquot of the pH-adjust solution. Add 10 mL of deionized        water and screw the cap tight. Shake/invert 5-8 times. Use        immediately; this mixture is single use only.    -   5) Slowly add the specified amount of antioxidant mixture to the        AAII using a stir bar and magnetic plate to incorporate. Allow        mixing until completely solubilized. Note any changes for        quality assurance records. (Optional: Pre-add the antioxidant        mixture to the AAII before freezing the initial lot of AAII.        Follow step 5 to incorporate into reagent.)    -   6) Adjust the pH to exactly 6.36 using the re-constituted        pH-adjust solution: add dropwise and do not overshoot. When        close to the desired pH value, allow at least 10 sec between        drops to ensure equilibrium is reached. A pH adjustment will        typically take 5-10 min and should not exceed 15 min.    -   7) Label the provided 24-well plates (when multiple tests of the        same type are performed at the same time, only one set of        standards plate is used).    -   8) Pipette 1.25 mL BB solution into the corresponding labeled        24-well-plates with a 12.5-mL Eppendorf Combitips (or        equivalent).    -   9) Pipette 1.25 mL of pH-adjusted (6.36) AAII solution into the        corresponding labeled 24-well plates with another 12.5-mL        Eppendorf Combitips.    -   10) If the substance is a solid with a pretest solubility check        greater than 0.350, or a liquid, place membrane discs into each        well (if solid, this should already have substance within it).        Carefully lift the 24-well plates and check the bottom to make        sure there are no bubbles between the contacting membrane and        solutions. Gently tap the side of the plate or lift the discs to        get rid of any bubbles.    -   11) Label each plate on the top and side using a Sharpie pen.        Optional: Label the lid of each 24-well plate as indicated above        and place the respective lid above the 24-well plates to be used        as a guide. For liquid samples, doses of 25 μL, 50 μL, 75 μL,        100 μL, and 125 μL will be used. For solid samples, doses of 50        mg, 100 mg, 150 mg, 200 mg, and 250 mg will be used.    -   12) Pipette 125 μL triplicates of standard 0 into three wells:        triplicate standard IV, triplicate of Standard III, and singles        of QC 1 and QC 2 into the appropriate wells.    -   13) Pipette 25, 50, 75, 100, and 125 μL of the unknown test        sample into the appropriate BB and AA wells.    -   14) Pipette 125 μL (or 250 mg if solid, pre-weighed) of the test        sample into the wells labeled TN+ and TNB.    -   15) Add 125 μL TN for liquid samples or 250 μL TN for solid        samples to the three wells on the left side labeled TN+, TN−,        and TNB (directly to center of well). If solid, mix substance        with pipette tip to ensure at least some of the solubilized test        substance is in contact with the membrane at the bottom of the        sample well.    -   16) Place each 24-well plate in container provided. Seal the        container by pressing firmly on all edges and then along the        entire edge of box. Replace damaged incubation boxes that do not        form good seal.    -   17) Place the entire container in a 30.6-31.3° C. incubator and        set a timer for 18 h. Incubation time should be 18-19 h (optimum        18 h, time is allowed when conducting multiple assays at the        same time).

Reading the 400 nm (a) Assay Results

-   -   1) Turn on the spectrophotometer for at least 10 min before use.    -   2) Adjust the spectrophotometer to 400 nm, absorbance mode.        Press “esc,” Press “General Tests,” Press “Basic ATC,” Press        “Set Wavelength,” enter “400,” and then press “enter.” If not in        absorbance mode, press “mode.” Adjust to absorbance mode.        Readout should include “A” at the end.    -   3) Blank the spectrophotometer: Add 1 mL BB solution to a        cuvette, place in the spectrophotometer, and push the blank        button. The spectrophotometer should read “0.000A” (+/−0.002).    -   4) After the assay incubation time is complete, remove the plate        from the incubator; remove the ocular discs from each well and        dispose of them appropriately. Check each disc for damage. Make        a record of damaged discs or any other abnormality (precipitate,        color, etc.). Fluid in the disc is normal; it is related to        denaturation via osmotic forces.    -   5) Mix the solution in each well individually with the tip of a        1-mL pipette prior to measuring the OD. This involves rapidly        and forcefully scraping the bottom of the well with a standard        size 1-mL pipette tip five times in one direction (in rapid        zig-zag pattern), scraping the tip around the bottom edge 2-3        times, and then repeating two more times (for a total of three)        to solubilize any white precipitate that has formed on the        bottom of the well of the 24-well plate. Mix with force and        attempt to hear an audible scraping sound (not possible with        some viscous fluid samples). The precipitate at the bottom may        not be visible. The precipitate at the bottom does not readily        go into solution. Forceful and complete mixing is required for        accurate results. After mixing, immediately aspirate the        solubilized solution from the center bottom of the well.    -   6) Immediately aspirate 500 μL into a test cuvette after mixing.        Tap the cuvette on the bench top to allow any bubbles to rise to        the surface.    -   7) Immediately measure the OD at 400 nm using a recently blanked        (with BB) spectrophotometer.    -   8) Include negative values on the data capture sheet.    -   9) Record the result and any notes on the data capture sheet.        (If readings fail to stabilize, consult the supplier for        additional procedural information).

Data Analysis

-   -   1) Sample OD values are recorded under the column “Sample OD”        for each respective concentration.    -   2) Blank OD values are recorded under the column “Blank OD” for        each respective concentration.    -   3) Measured values (MV=Net OD values) are obtained by        subtracting the Blank OD value and the average standard 0 OD        value from the Sample OD values for each respective        concentration. (Note: if less than zero, enter 0.)    -   4) Standard value (SV) is obtained by subtracting the average        standard 0 value from the average of the measured values for the        standards.    -   5) For each measured unknown test value (MV), assign a numerical        value (“score”) by using the closest standard value (CSV).    -   6) Identify the standard with the closest OD value (either        standard IV or III). Divide the measured OD by the closest        standard value (MV/CSV) and multiply this value by the CSV        designation (DV) (either IV=8.0 or III=12.5).    -   7) The resulting value=irritation score for the sample in        question. Populate the template with both OD values and        irritation scores.    -   8) Calculate the TN Value: Subtract both the TNB OD and the TN−        OD from the TN+ OD.    -   9) Calculate the irritant score for the TN following steps        above. If the TN score is the highest score, base the irritancy        prediction on the TN score using the prediction models.        Disregard negative TN Measured values and scores.    -   10) For the assay results (25-125 titrations) and the TN,        convert the calculated irritation scores into EPA and GHS        categories using the prediction models. Only the highest score        (including TN) is used for the final prediction.    -   11) The FP rate was significantly less when the antioxidant        mixture was used. The reduction of overpredicted nonirritants is        attributed to the ability of the antioxidant mixture to quench        the reactivity of nonirritant chemicals. 2,2-dimethyl-3-pentanol        CASRN 3970-62-5 (GHS NC, EPA III), 2-ethylhexylthioglycolate        CASRN 7659-86-1 (GHS NC, EPA IV), and iso-propyl bromide CASRN        75-26-3 (GHS NC, EPA IV) were all overpredicted, but were        corrected with the addition of the antioxidant mixture to        reflect the GHS classification of NC.

1.2 Additions to Tissue Culture Media for EPIOCULAR, STE, and OtherCell- or Tissue-Based Tests for Ocular Toxicity

To include the antioxidant mixture in cell-based assays, the mix can beadded to the medium or the test sample. Per the EPIOCULAR protocol byMatTek Corporation, the EpiOcular Assay Medium should be warmed toapproximately 37° C. The antioxidant mixture, containing one or more ofthe following: GSH, L-cysteine, L-tyrosine, ascorbic acid, and/or uricacid, is added to and solubilized in the medium until a homogenousmixture is reached. The tested range of ascorbic acid concentrationincluded in the EPIOCULAR test method was from 1,703.4 to 17,033.8 μM.Following this, 1.0 mL of Assay Medium is aliquoted into the appropriatewells of pre-labeled 6-well plates, the tissues should be removed fromthe 24-well plates, and the insert is then transferred into the 6-wellplates and preincubated in the Assay Medium (MatTek Corporation, 2021).Similarly, the antioxidant mixture can be applied to the media for theSTE test method (ICCVAM-NICEA™, 2013). The standard operating proceduresare listed as follows:

Modified EPIOCULAR—The following procedures were adapted from EPIOCULAREye Irritation Test (OCL-200-EIT) (2021) by MatTek Corporation(Available at:https://www.mattek.com/wp-content/uploads/OCL-200-EIT-Eye-Irritation-Test-Protocol-MK-24-007-0055_02_02_2021.pdf).Some procedures are from the MatTek EpiOcular Eye Irritation Test(OCL-200-EIT) Protocol. Additional new steps to the procedure areincluded as steps 2, 14, 15, 17, 18, 19.

Tissue Preincubation

-   -   1) RhCE tissues (purchased from MatTek Corporation) are        equilibrated for about 15 min.    -   2) The antioxidant is added to the Assay Medium and used for all        steps that follow and/or the antioxidant is added to the test        chemical or overlayed over the tissue prior to test chemical        exposure. Antioxidant procedure: 2.7 mg of the antioxidant        (Ascorbic acid, CASRN 50-81-7) was added to 10 mL of the        EpiOcular kit's media (OCL-100-ASY, Lot No. 021821TTC), mixed        with a stir bar for 5 minutes, and the pH was adjusted using        Sodium hydroxide (NaOH, CASRN 1310-73-2) or Hydrochloric acid        (HCl, CASRN 7647-01-0).    -   3) The DPBS was prepared in two beakers of 100 ml. In beaker A,        27.2 mg of Ascorbic acid was added, mixed with a stir bar for 5        minutes and the pH was adjusted to 7.35 using HCl or NaOH. In        beaker B, 20.1 mg of Ascorbic acid was added, mixed with a stir        bar for 5 minutes and the pH was adjusted to 7.37 using HCl or        NaOH.    -   4) Cell culture medium was warmed to approximately 37° C. and        one mL aliquoted into the appropriate wells of 6-well plates.    -   5) Tissues were removed from the shipping containers forceps and        then into the 6-well plates and incubated at standard culture        conditions for 1 h, then assayed medium changed and incubated        overnight (16-24 h).

Test Substance Exposure

-   -   1) Antioxidant procedure: During this Pre-Treatment step, 100 μL        of DPBS (without Ascorbic acid) was added to tissues of        Condition 1 (no Ascorbic acid added) and 100 μL of DPBS (with        Ascorbic acid) was added to tissues of Condition 2 (with        Ascorbic acid added).    -   2) Test Article Exposure: After the 30±2-min pretreatment with        and without ascorbic acid, each test is tested by applying 50 μL        topically. The tissues were then incubated at standard culture        conditions for 30±2 min.    -   3) Antioxidant procedure: The test article used was Styrene        (CASRN 100-42-5, Lot No. MKCM4502). 50 μL of styrene was added        to the tissue inserts of Conditions 1 (no Ascorbic acid added)        and 2 (with Ascorbic acid added).    -   4) Rinsing: At the end of the 30-min test chemical exposure        time, the test articles are removed by rinsing the tissues.    -   5) Antioxidant procedure: Tissue inserts of Condition 1 (no        Ascorbic acid added) were rinsed using DPBS (no Ascorbic acid        added) and tissue inserts of Condition 2 (with Ascorbic acid        added) were rinsed using DPBS (with Ascorbic acid added).    -   6) Antioxidant procedure: After rinsing, the tissue inserts of        Condition 1 (no Ascorbic acid added) were immersed into 5 mL of        the media without Ascorbic acid added and tissue inserts of        Condition 2 (with Ascorbic acid added) were immersed into 5 mL        of media for 60 minutes with Ascorbic acid added for 60 minutes.    -   7) Antioxidant procedure: After the 60 minutes of immersion, the        tissue inserts of Conditions 1 (no Ascorbic acid added) and 2        (with Ascorbic acid added) were removed and placed into the        6-well plate containing 1 mL of the corresponding media (with or        without Ascorbic acid) for another 60 minutes. The tested range        of ascorbic acid included in the EPIOCULAR test method is from        1,703.4 to 17,033.8 μM.

Modifications Required for Solids

-   -   1) Each solid is tested by applying one leveled volumetric        spoonful of material to be tested into tissue insert        (approximately 50 mg). and incubated for 6 hours.    -   2) Rinsing: At the end of the 6 hours, the test articles are        removed by rinsing (as above).    -   3) Post-treatment: After rinsing, the tissue inserts are        immersed in 5 mL of previously warmed Assay Medium    -   4) Recovery: Next, each insert is removed from the Assay Medium,        and transferred to a plate containing 1 mL of Medium. The        tissues are then incubated for 18 hours under cell culture.

Cell Viability Test

-   -   1) After the post-treatment incubation, the MTT viability test        is done.    -   2) A 1.0 mg/mL MTT solution is prepared and 300 μL of the MTT        solution is added to each well of a 24-well plate. each insert        is removed from the 6-well plate and placed into the 24-well        plate containing 0.3 mL of MTT solution. The plate is then        incubated for 3 hours under culture conditions.    -   3) After incubation, each insert is removed from the 24-well        plate and then transferred to a 24-well plate containing 2.0 mL        of isopropanol. The plates are sealed with saran wrap and are        either stored overnight with refrigeration. After this, tissue        inserts are pierced and the liquid is decanted into the well        from which it came.    -   4) The extract solution is mixed and two 0.2 mL aliquots from        each well transferred into a 96-well plate.    -   5) The absorbance at 570 nm (OD570) of each well is measured        with a plate reader.    -   6) If the MTT OD570 is 60% or greater of that generated by the        negative control, the material tested is classified as a        nonirritant. On the other hand, if the test substance OD570 is        less than 60% of the negative control, the test material is        considered an ocular irritant.

Alternative Visual Determination of Viability and Irritant Prediction

-   -   1) Since viable cells turn MTT purple, the relative viability of        one tissue to the next can be determined by a visual inspection.        The more purple the more alive the cells and the less irritating        the test substance applied to the cells are. For example, a        large area of white indicates a lot of cell death and an        irritating substance. On the other hand, a small area of white        or areas of color indicate less cell killing (higher cell        viability) and a less irritating test substance.    -   2) If the purple area is 60% or greater, the material tested is        a classified as a nonirritant. On the other hand, if the purples        area is less than 60% the test material is considered an ocular        irritant.    -   3) The addition of the antioxidant mixture into the tissue        medium has the potential to reverse the overprediction of the        known FPs that have been tested by EpiOcular. A real example is        provided for styrene, a current overpredicted irritant of the        EpiOcular test, suggests that the correct nonirritant        classification will result when the antioxidant mixture is        introduced into the cell-based assay. As shown in FIG. 3 , when        the above procedure was followed, application of the FP chemical        styrene resulted in extensive cell killing (condition 1,        indicated by the large area of white, where there are no viable        cells). However, when ascorbic acid (0.3 mg/ml) was added to the        PBS overlay and culture medium, there was significantly less        cell killing (Condition 2, area of dark, showing viable cells),        and a prediction of nonirritant (TN). This indicates that the        addition of the antioxidant results in a reduction of the rate        of false positives and an increase in test method specificity        and accuracy.    -   4) In summary, results for the novel addition of antioxidants        described above, are shown in FIG. 3 . The viability of cells is        observed or quantified using MTT        (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)        treatment, which is a dye that forms a blue/purple color when        reduced (through continued redox cycling of living cells). In        Repeat 1 and Repeat 2 of the “No Antioxidant” condition        (Condition 1) on the left side, there is a visually clear region        of dead cells after treatment with Styrene. There is no color in        the region of dead cells between MTT is not reduced. In this        condition, no Ascorbic acid was added to the DPBS or media when        following the antioxidant procedure. In Repeat 1 and 2 of the        “Antioxidant” condition (Condition 2) on the right side, there        is a region of live cells after treatment with Styrene. The        region of live cells is a blue/purple color which indicates that        MTT is being reduced and the cells are viable. The only        difference is, in this condition, an antioxidant was added to        the DPBS and the media. Overall, based on the results and the        clear distinction between the region of dead cells and region of        lives cells in both conditions, it can be concluded that the        antioxidant procedure with this RhCE procedure produced a        significant increase in viability, and therefore, the false        positive styrene, is no longer a false positive when        antioxidants are added. This result is consistent with the        results for the addition of antioxidants to the OptiSafe test.

Using a Buffered High Concentration Ascorbic Acid Formulation forPreventing Corneal Damage Following Exposure to Irritant—Modeled in theModified EPIOCULAR Test System

FIG. 4 depicts results from the modified EPIOCULAR reconstituted humancorneal epithelium test system (details of assay above) in which a highconcentration antioxidant in a buffered salt solution was added. Afterexposure to known irritants (D, E, F) or nonirritant controls (A, B, C)transiently to mimic an accidental exposure into the eye, the oculartissues were washed with the buffered salt solution without ascorbicacid (left column) or with a high concentration (17.0 mM; 3 mg/ml)ascorbic acid (right column). The dark staining is associated withviable cells in the corneal epithelium—i.e., greater protection againstdamage. This experiment demonstrates what would be expected when anocular irritant contacts the eye in vivo, and the eye is thenwashed/flushed as a first aid measure to reduce damage to the eyetissue. The addition of the high concentration ascorbic acid to thebuffered salt solution wash significantly reduced the corneal damage(evidenced by higher cell viability—dark staining) caused by knownirritants, compared to the cellular damage (evidenced by extensive celldeath—very little staining) in reconstituted corneal tissue washed withthe same buffered salt solution without any added antioxidant; comparethe dark stained areas (viable cells) seen in the ascorbic acid groupwith the light, translucent areas (dead cells) seen in the group withoutantioxidant. Death of eye tissue is associated with irritation and/orscarring and permanent eye damage. Thus, buffered high concentrationantioxidant appears to be highly effective in mitigating corneal damagecaused by chemical irritants.

STE—The procedures are available from the NICEA™ Review Document (2013)Short Time Exposure (STE) Test Method Summary Review Document (Availableat:https://ntp.niehs.nih.gov/iccvam/docs/ocutox_docs/ste-srd-niceatm-508.pdf).Additional steps are included as steps 1 and 2.

-   -   1) Prepare Medium. Add a specified amount of the antioxidant        mixture to the media and allow to completely solubilize. For        best results, use a stir bar and magnetic plate to slowly stir.        Once the homogenous solution is obtained, proceed using medium        with the incorporated antioxidant mixture through the assay.        Alternatively, add the antioxidant to the chemical to be tested,        or overlay the tissue with antioxidant medium prior to test        chemical exposure.    -   2) Alternatively, add the antioxidant to the chemical to be        tested or overlay the tissue with antioxidant medium prior to        test chemical exposure. Start by preparing the media (MEM) and        add a specific amount of the antioxidant mixture to the media        and allow it to completely solubilize. For the best results, use        a stir bar and a magnetic plate to slowly stir until homogenous.        If necessary, adjust the pH using Sodium hydroxide (NaOH, CASRN        1310-73-2) or Hydrochloric acid (HCl, CASRN 7647-01-0). Once the        homogenous solution is obtained, proceed using MEM with the        incorporated antioxidant mixture throughout the entirety of the        assay.    -   3) The inclusion of the antioxidant mixture in the media for STE        allows for a reduction of FP chemicals predicated by the        cell-based assay. In the prophetic analysis of styrene, a GHS        NC, the novel addition to the STE assay is projected to        reclassify this chemical. The antioxidant mixture is indicated        to increase the measured cell viability to correctly categorize        ethyl acetate as a TN.

1.3 Adding to Media for BCOP, ICE, IRE (Isolated Rabbit Eye), and anyOther Ex Vivo Eye Test System, Including Human

Addition of the antioxidant mixture for ex vivo assays such as BCOP,ICE, IRE, etc. can be to the media or substance to be tested. The finalantioxidant mixture includes one or more of the following: GSH,L-cysteine, L-tyrosine, ascorbic acid, and/or uric acid. For BCOP, thepreparation of the corneas entails exposing them to a medium of MEM.Prior to mounting the corneas, the antioxidant mixture will be added tothe chemical to be tested or overlayed on the eye before addition ofchemical or be added to the culture medium. The antioxidant can beincorporated into the ICE test method using the saline solution (OECD,2018). The procedures are listed as follows:

1.4 Additions to Eggs for HET-CAM or CAMVA

To include the antioxidant mixture in egg-based assays, the mix can beadded to the overlay or the test sample. After the exposure of thevessels of the membrane, the antioxidant mixture can be added torepresent the mechanistic defenses in vivo that are not currentlyaccounted for in egg-based assays (ICCVAM, 2010). The HET-CAM procedureis described by the ICCVAM-Recommended Test Method Protocol: Hen's EggTest-Chorioallantoic Membrane (HET-CAM) Test Method (2010) (availableat:https://ntp.niehs.nih.gov/iccvam/docs/protocols/ivocular-hetcam.pdf).

-   -   1) Add the antioxidant mixture to the surface of the membrane.        Ensure the entirety of the membrane is covered in the        antioxidant mixture. Leave the mixture on while applying the        test substance.    -   2) Alternatively, add the antioxidant mixture to the chemical to        be tested or the surface of the membrane. Start by preparing the        antioxidant mixture and by adding the specific amount of        antioxidant to a PBS solution and allow it to completely        solubilize. For the best results, use a stir bar and a magnetic        plate to slow stir until homogenous. If necessary, adjust the pH        using Sodium hydroxide (NaOH, CASRN 1310-73-2) or Hydrochloric        acid (HCl, CASRN 7647-01-0). Once the homogenous solution is        obtained, proceed using this antioxidant mixture throughout the        entirety of the assay. When applying the antioxidant mixture to        the surface of the membrane, ensure the entirety of the membrane        is covered    -   3) A prophetic analysis of styrene, a GHS NC and overpredicted        FP by the HET-CAM test method, indicates that the HET-CAM score        would fall below the irritant cut-off, resulting in a TN        prediction of a GHS NC, with the use of the antioxidant mixture.

Depth of Injury (DOI) (Lebrun Labs LLC Procedure)

Only food source eyes are suitable for this procedure. Ensure properdocumentation that the eyes are extra eyes from a food processingfacility before placing the order. The day before the eyes arrive, placeone bottle of antioxidant medium and one bottle of 1×PBS antioxidantbuffer solution (Ascorbic Acid; 1.70 mM (0.3 mg/ml)) into the incubatorand place one bottle of 1×PBS antioxidant buffer solution (AscorbicAcid; 1.70 mM (0.3 mg/ml)) into the refrigerator.

Receiving the Eyes

-   -   1) Remove packing slip from the box and fill out the receiving        form with the necessary information.    -   2) Retrieve a large Pyrex dish and place one ice pack in it.    -   3) Open the box and open the Styrofoam box that contain the eyes        and take out the bag containing the eyes in jars. Remove the        jars and immediately place on the ice packs in the large Pyrex        dish prepared.

Eyelid Removal

-   -   1) Place a large piece of gauze on the plastic-lined pad in the        work area.    -   2) Remove one eye from the jar and place on the gauze.    -   3) Use the scissors to make an incision behind the eyelids where        it meets the sclera and cut until the whole eyelid is removed        from the eyeball.    -   4) Use the scissors to cut the connective tissue, following the        curved shape of the cornea but without touching it. Note: Do not        remove too much connective tissue or it will be difficult to        hold the eye when rinsing after dosing.    -   5) Place the eye in the “good” eye jar with refrigerated 1×PBS        (Jar #1).    -   6) Continue until all eyes have the eyelids and connective        tissue removed.    -   7) Place an eye holder in a small Pyrex dish.    -   8) Using a plastic pipette, fill the eye holder with Lissamine        green dye.    -   9) Take the 18-gauge needle and syringe and remove the needle to        aspirate the refrigerated 1×PBS.    -   10) Take an eye and dip the cornea into the Lissamine green dye        in the eye holder and slowly move the eye around to ensure the        cornea is completely covered.    -   11) Using the 18-gauge needle and syringe filled with the        refrigerated 1×PBS, wash the Lissamine green dye off over the        waste container.    -   12) Inspect the corneas for any Lissamine green dye indicating        damage.    -   13) Place the undamaged eyes in a new jar (Jar #2) filled with        fresh refrigerated 1×PBS and damaged eyes in a separate        container to discard.

Preincubation

-   -   1) Open the 12 well plate(s) and fill each well with a “good”        eye ensuring the cornea is facing up and the optic nerve is at        the bottom of the well.    -   2) Add varying amounts of warm media from the incubator to each        well ensuring the limbus is not covered. Note: The amount will        vary per well due to inconsistent sizes of the eyes.    -   3) Put the lid onto the 12 well plate(s) and place into the        incubator (37° C., 5% CO2) for one hour.

Dosing

-   -   1) After incubation, remove the 12 well plate(s) from the        incubator and place into the hood.    -   2) Label the wells on the lid with what is being tested.    -   3) Place an eye on the holder and carefully place a new cloning        ring in the middle of the cornea without scratching it.    -   4) Fill the syringe with 40 mL of refrigerated 1×PBS antioxidant        buffer solution (Ascorbic Acid; 1.70 mM (0.3 mg/ml)) by sucking        it up from the beaker    -   5) Put the needle back on the syringe by twisting on, and then        remove the cap that protects the needle.    -   6) Place the syringe flat on a sterile piece of foil.    -   7) Set the timer for “counting up”.    -   8) Add by micropipette first 100 μL of antioxidant buffer        solution (Ascorbic Acid; 1.70 mM (0.3 mg/ml)) then 100 μL of        test substance    -   9) Start the timer for one minute.    -   10) After the one minute is over, remove the dosing ring and put        in the proper container, and pick up the tissue without touching        the cornea and hold it over the container for liquid waste.

Washing and Postincubation

-   -   1) Directly dispense 20 mL of antioxidant buffer solution        (Ascorbic Acid; 1.70 mM (0.3 mg/ml)) onto the area of the cornea        that was exposed to the sample as well as the back of the        eyeball to ensure all the sample is washed off.    -   2) Place the eye back into its original well.    -   3) Continue for the rest of the eyes and samples.    -   4) Once all eyes are dosed, label a new 12 well plate and        transfer the eyes into it.    -   5) Then add fresh media to the new 12 well plate(s).    -   6) Place the 12 well plate(s) into the incubator at 37° C.+5%        CO2 for 24 hours.

Cornea Extraction and Fixation

-   -   1) Retrieve the correct number of 12 well plate(s) and label the        lid above each well with the correct dosing test substance.    -   2) Fill each well with 5 mL of the 4% paraformaldehyde    -   3) Once the 24-hour incubation is complete, remove the plate(s)        from the incubator and place near cutting area.    -   4) Remove an eye and hold using the 2×2 gauze with the cornea        facing up and ensuring the gauze does not cover the cornea or        limbus.    -   5) Using the scalpel, poke a hole in the sclera about 2 mm from        the cornea.    -   6) Using the scissors, cut along the edge of the cornea ensuring        a 2 mm border of sclera.    -   7) Make sure the cuts are smooth and ensure the cornea does not        come into contact with the scissors or the cut mat.    -   8) Once the cornea is separated from the rest of the eye, place        the cornea with the iris attached directly into the        paraformaldehyde and slowly move back and forth to get rid of        any folding.    -   9) Continue until all corneas have been removed.    -   10) Saran Wrap the plate and then put the lid on    -   11) Put the plate(s) in the fridge.

Sucrose Infiltration

-   -   1) Label a new 12 well plate.    -   2) Remove the plate(s) with the corneas suspended in 4%        paraformaldehyde from the fridge.    -   3) Place a cutting mat on the bench and a large gauze pad down.    -   4) Remove each cornea one by one and use the small forceps to        remove the iris.    -   5) Cut the cornea in half.    -   6) Once the cornea is cut in half, place the cornea back into        the correct well of paraformaldehyde.    -   7) Get a new 12 well plate and fill each well with 5 mL of 10%        sucrose solution.    -   8) Transfer the corneas from the 4% paraformaldehyde solution        into the new 12 well plate containing the 10% sucrose.    -   9) Let sit for 15 minutes.    -   10) During the 15 minutes, prepare the 2:1 10% sucrose:30%        sucrose solution in a 50 mL conical tube.    -   11) Once the 15 minutes is done, use a serological pipette to        remove the 10% sucrose from the wells.    -   12) Fill the same wells with 4.5 mL of the 2:1 10% sucrose:30%        sucrose solution.    -   13) Let sit for 15 minutes.    -   14) During the 15 minutes, prepare the 1:1 10% sucrose:30%        sucrose solution.    -   15) Once the 15 minutes is done, use a serological pipette to        remove the 2:1 10% sucrose:30% sucrose solution from the wells.    -   16) Fill the same wells with 5 mL of the 1:1 10% sucrose:30%        sucrose solution.    -   17) Let sit for 15 minutes.    -   18) During the 15 minutes, prepare the 1:2 10% sucrose:30%        sucrose solution.    -   19) Once the 15 minutes is done, use a serological pipette to        remove the 1:1 10% sucrose:30% sucrose solution from the wells.    -   20) Fill the same wells with 4.5 mL of the 1:2 10% sucrose:30%        sucrose solution.    -   21) Let sit for 15 minutes.    -   22) Once the 15 minutes is done, remove the 1:2 10% sucrose:30%        sucrose solution from the wells.    -   23) Fill the same wells with 5 mL of 30% sucrose (#1).    -   24) Let sit for 15 minutes.    -   25) Once the 15 minutes is done remove the 30% sucrose solution        and replace it with another 5 mL of 30% sucrose solution (#2).    -   26) Let sit for 15 minutes.    -   27) Once the 15 minutes is done remove the 30% sucrose solution        and replace it with another 5 mL of 30% sucrose solution (#3).    -   28) Let sit for 15 minutes.

OCT Embedding and Liquid Nitrogen Freezing

-   -   1) Label all pink bags with your name, date, sample name, and        Lot #.    -   2) Label 1 large white bag with name, date, samples and # of        each, and Lot #.    -   3) Take out the correct number of Tissue-Tek cryomolds, and        forceps.    -   4) Fill the first square of the Tissue-Tek cryomold with OCT.    -   5) Grab the outer round edge of the cornea with the forceps and        place the straight edge into the OCT so it lays along the bottom        of the cryomold.    -   6) Ensure the cornea is completely vertical and not bent.    -   7) Inspect from above and the sides to ensure it is in the        correct orientation.    -   8) Rest the cryomold on the coat hanger holder and slowly insert        the cryomold into the Dewar Flask until only the bottom of the        cryomold is exposed to the liquid nitrogen.    -   9) DO NOT LET THE LIQUID NITROGEN TOUCH THE OCT DIRECTLY.    -   10) Once the bottom of the cryomold is in the liquid nitrogen,        you should be able to see the OCT from above go from clear to        completely white.    -   11) Once the OCT above the liquid nitrogen is completely white        and frozen and has no air bubbles, the entire cryomold can be        submerged into the liquid nitrogen and kept there for 20 seconds        to ensure adequate freezing.    -   12) After the 20 seconds is finished, use the coat hanger mold        to remove the cryomold and ensure all excess liquid nitrogen is        poured into the Dewar Flask.    -   13) The entire mold can then be placed in a Pyrex dish for a        couple of seconds.    -   14) Immediately transfer the entire cryomold with the frozen        cornea in it, to its respective labeled pink bag.    -   15) Immediately place the pink bag into the large white bag in        the −80° C. freezer for storage.

Cryosectioning

-   -   1) Lay out paper towel to put the slides on.    -   2) Label the slides using the following labeling system:    -   3) Once all slides are labelled, lay them out on the paper towel        in an organized manner. 4) Turn the light on in the cryostat.    -   5) Ensure the cryostat is at −25° C.    -   6) Ensure the small chuck is in the cryostat and in the proper        freezing area on the left.    -   7) Ensure the 1 g metal weight is in the cryostat and at the        correct temperature (if not in the cryostat, place the weight in        the cryostat and wait for it to cool down to the −25° C.        temperature).    -   8) Put the desired paint brush in the cryostat to make sure it        gets to the proper temperature.    -   9) Retrieve a bottle of OCT if not on top of the cryostat        already.    -   10) Check the micron setting to make sure it is set to the        correct micron value.    -   11) Retrieve the first sample from the −80° C. freezer.    -   12) Take the frozen sample (still in the Tissue-Tek cryomold)        and remove it from the pink bag.    -   13) Place the pink bag in the cryostat and the sample on top of        the pink bag.    -   14) Close the cryostat lid and leave the sample until it is able        to be popped out of the mold with ease (approximately 5-8        minutes).    -   15) When the sample is ready to be popped out, flip the mold        upside down so when the sample is popped out it will land        directly on the pink bag. The top of the frozen sample (the part        exposed to air while in the mold) should be touching the pink        bag. The bottom of the frozen sample (the part touching the        inside of the mold which has the flat side of the cornea) should        be facing upwards so you can slightly see the outline of the        sample.    -   16) Once the sample is popped out of the mold, leave it in the        orientation.    -   17) Get the OCT and squeeze it onto the chuck, starting from the        center and dispensing in a circular motion following the rings        on the chuck until the entire chuck has a layer of OCT on it.    -   18) Quickly pick up the frozen sample and place it on the OCT.    -   19) Get the 1 g metal weight and carefully place it on top of        the frozen sample so it adheres to the OCT and freezes        completely horizontal and flat to allow for proper cutting.    -   20) Close the lid and wait for the OCT to completely freeze and        bond to the frozen sample (approximately 5 minutes).    -   21) Once the OCT is completely frozen and the sample is ready to        be cut, open the lid and remove the 1 g weight.    -   22) Lift the chuck up and insert it into the block and tighten        to secure it.    -   23) Use the buttons on the left-hand side to adjust the distance        of the sample from the blade.    -   24) Once the sample is in the correct position/distance from the        blade, slowly turn the handle to see if further adjustment is        needed.    -   25) In the beginning, only certain parts of the sample may be        getting cut (like the middle, bottom, or edge)—this is normal.    -   26) Continue slowly turning the handle to trim the frozen sample        and eventually trim to the point where the entire frozen sample        is being cut evenly. As sections (full or partial) are being        cut, lift the glass and use the paint brush to wipe away the        excess to have a clean slate for future sections.    -   27) Once the sections being cut are full squares and the entire        cornea is being included in a section, it is ready to be put on        slides.    -   28) Use the paint brush to wipe away any sections and start with        a clean area.    -   29) Turn the handle to get a clean full section.    -   30) Lift the glass to expose the section. If the section is        slightly curled or scrunched, use the paint brush to straighten        it out by lightly putting the edge of the bristles on the bottom        part of the section and gently pulling towards yourself to        remove the wrinkles.    -   31) Get the slide and flip it over so the part where the section        sticks is facing down.    -   32) Hover the slide over the section in the exact spot it will        be placed on the slide.    -   33) Slowly put the bottom edge of the slide (closest to you) on        the bottom of the section and quickly roll the slide towards the        top of the slide (closest to the blade) to ensure the section        gets placed on the slide without air bubbles or folds.    -   34) Once the section is attached to the slide, flip it over so        the side that the section is on is faced up.    -   35) Place the slide back on the paper towel.    -   36) Put the glass back down gently so you can continue cutting        more sections.    -   37) Continue cutting sections one by one and adding them to each        slide closest to the plus signs.    -   38) Once all slides of the sample have 1 section on it, trim the        sections about 100 microns by turning the handle 15 times.    -   39) Use the paint brush to wipe away the sections and provide a        clean area.    -   40) Cut sections one by one again and add them to the middle of        the slide until each slide for the sample has a middle section        on it.    -   41) Trim the section again by 100 microns and use the paint        brush to wipe away the sections.    -   42) Cut sections one by one again and add them to the top of the        slide until each slide for the sample has a top section on it.        Each slide should have 3 sections on it now.    -   43) Once all slides have 3 sections on them the cutting is done.    -   44) Remove the chuck from the block and leave it outside of the        cryostat (approximately 1 minute) to slightly thaw the sample so        it can be removed from the chuck.    -   45) Once the sample has been slightly thawed, remove it from the        chuck and place it back into the pink bag and then the −80° C.    -   46) Either stain the slides in the same day, or sore the slides        in a slide box. Slide box must be properly labeled using        labeling tape regardless of same day staining or not.

Staining

-   -   1) Lay out the slides, face up, in the slide box.    -   2) So that the top and bottom edge (white writing part and plus        sign part) are laying on the groove where the slides would be        put into.    -   3) If the slides came from the fridge, let them get to room        temperature (approximately 10 minutes).    -   4) Put the Phalloidin and DAPI stock solution tubes on the        cooling rack and keep them covered with foil to protect them        from light.    -   5) Prepare the phalloidin mixture (this is enough to cover 16        sections). Dispense 800 μL of 1×PBS into the microcentrifuge        tube. Dispense 20 μL of phalloidin into the 1×PBS. Invert to mix        and place the conical tube in the cooling rack (covered with        foil) until ready to dispense the solution on the sections.        Phalloidin is light sensitive so keep covered at all times.    -   6) Dispense 50 μL on each individual cornea section.    -   7) Once all sections are covered with the Phalloidin solution,        close the lid of the slide box but do not completely close        it—just let the lid lay closed.    -   8) Get the foil and cover the slide box to prevent any light        from going through.    -   9) Turn off the lights in the room to help with light issues.    -   10) Let the solution sit for 20 minutes.    -   11) Once the 20 minutes is done, remove the foil and open the        lid.    -   12) Take each slide one by one and dump the phalloidin liquid in        the waste beaker.    -   13) Once the excess phalloidin has been removed, use a        disposable pipette to rinse the slide off with 1×PBS. Dispense        the 1×PBS at the top of the slide and let the PBS run down the        slide to rinse it. DO NOT dispense the PBS directly on the        sections or they could come off or get ruined.    -   14) Once all slides have been rinsed, lay them back in the slide        box and lay the lid closed so the DAPI solution can be made.    -   15) Prepare the DAPI mixture (this is enough to fill 1 conical        tube that holds 5 slides). Fill the 50 mL conical tube with 50        mL of 1×PBS. Dispense 10 uL of DAPI into the conical tube. Put        the lid on and shake until it is all incorporated. DAPI is light        sensitive so keep covered at all times.    -   16) Pour the DAPI solution into the Coplin jar and transfer the        slides into the jar.    -   17) Cover the Coplin jar with foil and turn the lights off once        again.    -   18) Keep the slides in the solution for 5 minutes.    -   19) Once the 5 minutes is up, take the slides out one by one and        use a disposable pipette to rinse off the excess DAPI solution        with 1×PBS.    -   20) Once the slides have been rinsed off, place them lying flat        in the slide box once again.

Cover Slip Addition

-   -   1) Place 2 stacked Whatman paper on the table and keep another        single paper on the side out of the way.    -   2) Dispense about 3 drops of glycerol on each individual cornea        section.    -   3) Slowly place a cover slip onto the slide starting from one        end and slowly angling the cover slip even more until the entire        thing is on the slide. Dropping the cover slip fast will cause        the formation of air bubbles and may ruin the section due to        potential movement.    -   4) Once the cover slip is completely on, pick up the slide and        place the side on its side at about a 45-degree angle on the        paper (slide is facing the paper and the cover slip side is        facing towards you).    -   5) Slowly increase the angle of the slide by bringing the top        side towards you until it makes a 90-degree angle with the        Whatman paper.    -   6) This will make a seal between the Whatman paper and the slide        and cause the excess 50% glycerol to be absorbed by the Whatman        paper.    -   7) Once the excess 50% glycerol is removed, flip the slides over        to the other side and hold it against the Whatman paper at a        90-degree angle to remove any excess 50% glycerol from that end.    -   8) Lay the slide on the slide box again face up and use the        Whatman paper left on the side to absorb any excess 50% glycerol        at the edges by carefully holding the edge of the paper to the        edge of the coverslip on the slide.    -   9) Once all of the excess 50% glycerol has been removed, the        slides are ready for imaging. 10) Keep them in the slide box        with the lid closed to prevent unnecessary light exposure.

Imaging

-   -   1) Turn on the microscope (black switch on the right side).    -   2) At the base of the microscope the word “set” will show up in        red indicating the microscope is on.    -   3) Turn on the fluorescence light on the power box to the left        of the microscope. 4) Give the light about 3 minutes to warm up.    -   5) While the light is warming up, click on the “DoI Images”        folder on the desktop.    -   6) Create a new folder within labeled “Experiment dd-mm-yy        Intact Cornea 24 hour Series”    -   7) Within that folder, make a folder for each sample that was        tested.    -   8) Within the folder for each sample make two folders each        labeled “100×” and “400×”    -   9) Open the Thor Camera software and click the down arrow on the        left of the screen that pops up.    -   10) Click “CS235MU”    -   11) Once the imaging window comes up, maximize the screen.    -   12) Put a slide in the microscope and align the 10× lens.    -   13) Under the eye piece there is a silver disc embedded into the        microscope that can rotate to change the fluorescence filters (4        settings labeled—1, 2, 3, & 4). 3-FITC-DAPI    -   14) Rotate the disc to filter 3 and look through the eye        piece—the cornea should be illuminated green with the epi being        brighter green than the stroma. This means the phalloidin        stained the cornea adequately.    -   15) Rotate the disc to filter 4 and look through the eye        piece—the cornea should be illuminated blue with the nuclei in        the epi a defined blue. This means the DAPI stained the cornea        adequately.    -   16) Now that the stain has been checked it is ready to be        imaged.    -   17) Keep the disc that controls the filter at number.    -   18) Starting with DAPI makes it easier and faster to focus the        image.    -   19) Ensure the 10× lens is being used.    -   20) While looking through the eye piece, position the cornea        where you want capture the image making sure to include the        epithelium, stroma, and endothelium. The cornea must be centered        in the screen and completely straight. Horizontal or vertical        depending on the orientation of the camera.    -   21) Once the cornea is set up in the correct position, pull out        the metal bar out to the left of the eyepiece on the side of the        microscope. Below the bar it will say “photo, photo/vis, vis”        This will allow the camera to see the image instead of you        through the eye piece.    -   22) At the top bar, click the icon that looks like 3 gears        (settings button). Move the settings window to the very right so        it does not block the screen where the image will be displayed.    -   23) Set the exposure time to 250 ms and then press enter.    -   24) In the top left corner click the round green button with the        white triangle in it. “Live image”    -   25) Once the image is displayed on the screen, adjust it up/down        left/right to make it centered on the screen.    -   26) If the image needs to be slightly rotated to make it        horizontal, slowly rotate the stage.    -   27) Once the image in centered and positioned correctly, use the        fine tune knob to focus the image until it is clear.    -   28) Once the image is clear, determine if the exposure time is        high enough or if it needs to be adjusted. For DAPI, the nuclei        should be visible and defined but not extremely bright.    -   29) Once the exposure is adjusted to the correct level, press        the live image button once again so it stops showing the live        image. The button should be a green circle with a white square        in it.    -   30) Then press the camera icon to take a picture.    -   31) The camera icon and the icons near it will grey out for a        little bit and then the color will come back.    -   32) Once the color comes back to the icons, the picture is done        being taken and ready to save.    -   33) To save the image, press the floppy disk with the tree in        the bottom right.    -   34) It will bring up the file explorer where you find the folder        you made earlier and save it under the correct sample name. This        will be saved under the 100× folder.    -   35) The picture will be labeled: “‘sample name’ ‘slide #’        ‘section letter’ 100×‘DAPI or FITC’” Ex. “Water 1A 100×DAPI”    -   36) Once the DAPI image is saved under the 100× folder, the        phalloidin image is ready to be taken.    -   37) DO NOT MOVE THE STAGE.    -   38) ONLY turn the disc to change the filter from number 4 to        number 3 so the phalloidin stain can be seen.    -   39) Once the filter is on number 3, click the settings button        once again.    -   40) Start off by changing the exposure to approximately 5000.    -   41) Click the live image button and see how bright the        phalloidin stain is.    -   42) The goal is to have the stroma bright and clearly visible.        If the epi will become over exposed if done correctly and that        is acceptable.    -   43) Continue to adjust the exposure until the stroma is very        bright and clear.    -   44) Once the exposure is adjusted and the image is focused, stop        the live image display.    -   45) Click the camera icon and wait for the image to be taken.    -   46) Save the image in the same folder and label it correctly (in        this case it will be the same label as the previous picture but        will have “DAPI” switched out for “FITC”): “‘sample name’ ‘slide        #’ ‘section letter’ 100×‘DAPI or FITC’” Ex. “Water 1A 100×FITC”    -   47) Continue taking pictures for the rest of the sections on the        slide until all 100× images are complete. Between each section,        push the rod in so you can see the sections through the eye        piece. When ready to image, pull the rod out again so the camera        can capture the image.    -   48) Turn the lens to the 40× lens.    -   49) Turn the disc so the filter is on number 4 (DAPI).    -   50) Push the rod in so you can visualize the sections through        the eye piece.    -   51) Position the light over the first section and find a        straight clear section of the epithelium.    -   52) Pull the rod out.    -   53) Set the exposure to 150 ms and press enter.    -   54) Press the live image button and see if the image is at the        correct exposure. The nuclei should not be too dark or too        bright.    -   55) Once the image has the correct exposure and is focused, stop        the live image.    -   56) Press the camera icon and wait for the image to be taken.    -   57) Press the save image button and save the image under the        correct sample name and in the 400× folder.    -   58) Label it correctly: “‘sample name’ ‘slide #’ ‘section        letter’ 400×‘DAPI or FITC’” Ex. “Water 1A 400×DAPI”    -   59) Once the DAPI image is saved under the 400× folder, the        phalloidin image is ready to be taken.    -   60) Do not move the stage.    -   61) Only turn the disc to change the filter from number 4 to        number 3 so the phalloidin stain can be seen.    -   62) Once the filter is on number 3, click the settings button        once again.    -   63) Start off by changing the exposure to approximately 2000.    -   64) Click the live image button and see how bright the        phalloidin stain is.    -   65) The epithelium should be illuminated but not over exposed.        It should be clear and defined.    -   66) Once the exposure is adjusted and the image is focused, stop        the live image.    -   67) Press the camera icon and wait for the image to be taken.    -   68) Save the image in the same folder and label it correctly (in        this case it will be the same label as the previous picture but        will have “DAPI” switched out for “FITC”): “‘sample name’ ‘slide        #’ ‘section letter’ 400×‘DAPI or FITC’”. Ex. “Water 1A        400×FITC”.    -   69) Continue taking pictures for the rest of the sections on the        slide until all 400× images are complete. Between each section,        push the rod in so you can see the sections through the eye        piece. When ready to image, pull the rod out again so the camera        can capture the image.

The results shown in Table 12 indicate that the false positive rate issignificantly reduced by the presence of antioxidant formulation for theDoI procedure compared with other test methods.

TABLE 12 in vivo BCOP BCOP Chemical Name CASRN GHS (LLBO) (OP-KIT) EPIOI Do Sodium lauryl sulfate (3%) 151-21-3 NC FP FP FP FP TN Ethylacetate 141-78-6 NC FP FP FP FP Cyclohexanone 108-94-1 NC FP FP FP2-(2-Ethoxyethoxy) ethanol 111-90-0 NC FP FP TN 3-Phenoxy benzyl alcohol13826- NC FP FP FP 35-2 2-Ethoxyethyl methacrylate 2370-63- NC FP FP TN0 2,4-Pentanedione 123-54-6 NC FP FP FP 1-Nitropropane 108-03-2 NC FP FPTN Ethylene glycol diethyl ether 629-14-1 NC FP FP FP Styrene 100-42-5NC FP TN FP 1,9-Decadiene 1647-16- NC FP TN TN TN 1 p-Methylthiobenzaldehyde 3446-89- NC TN TN 7 2,2-Dimethyl-3-pentanol 3970-62- NCFP FP FP 5 1,3-Di-iso-propylbenzene 99-62-7 NC FP TN FP TN Triphenylphosphite 101-02-0 NC FP TN Triethylene glycol 112-27-6 NC FP TN 0 TN 0TN 3 TN 2 TN 9 TN 4 FP 5F P 12 FP 8 FP 7 FP FPR 100% 100% 80% 80% 43.8%(4/4) (5/5) (12/15) (8/10) (7/16) Legend: GHS = Globally HarmonizedSystem of classification and labeling of chemicals; NC = Nonclassified;BCOP (LLBO)/(OP-KIT) = Bovine Corneal Opacity & Permeability LaserLight-Based Opacitometer/Opacitometer Kit; EPI = EpiOcular; Ol = OcularIrritection; Dol = Depth of Injury; CNM = Criteria not met; FP = Falsepositive; TN = True negative.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.

The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

One skilled in the art will appreciate that this and other processes andmethods may be implemented in differing order. Furthermore, the outlinedsteps and operations are only provided as examples, and some of thesteps and operations may be optional, combined into fewer steps andoperations, or expanded into additional steps and operations withoutdetracting from the essence of the disclosed embodiments.

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What is claimed is:
 1. A method for reducing false positive rates of nonanimal eye irritation tests, the method comprising: overlaying an antioxidant formulation onto a surface of a differentiated eye tissue, comprising reconstituted human corneal epithelium or excised eye tissue; adding a test substance to the antioxidant formulation on the surface of the differentiated eye tissue; exposing the differentiated eye tissue to the test substance for a first period of time; washing the surface of the differentiated eye tissue with a buffered salt solution to remove the test substance; after a second period of time, measuring cell viability of the differentiated eye tissue; and relating the measured cell viability to an index of irritation, which can be categorized according to established ocular irritancy classes, wherein the false positive rate is reduced compared to performing the method without overlaying with the antioxidant formulation.
 2. The method of claim 1, wherein the established ocular irritancy classes are selected from a nonirritant, a minimal irritant, a mild irritant, and a severe irritant.
 3. The method of claim 1, wherein the established ocular irritancy classes include GHS categories NC, 2, 2B, 2A, and 1, or EPA categories IV, III, II, and I.
 4. The method of claim 1, wherein the antioxidant formulation comprises ascorbic acid.
 5. The method of claim 1, wherein washing further comprises a subsequent wash with additional antioxidant formulation. 